I am not aware of any organic specific molecule that inhibits CNG channels and I would love to hear of one if anybody knows. Meanwhile, I know of Inorganic blockers that have been used to block animal as well as plant CNGs

1/ Nickel seems to block animal CNGs (try 50-100 microM). Lower concentrations (like 10 microM) might increase CNG open probability (see the review by Zagotta and Siegelbaum, 1996).

2/ Also increasing intracellular Calcium (to say 2-3 microM) is sufficient to dampen CNG Channel activity (see Zufall et al., 1991)

3/ Plant CNGCs are blocked by Gadolinium and Lanthanum. Gd3+ is more potent in my hands. I use Gd3+ at 50 to 100 microM but in your case, I would start at lower concentrations (5-10 microM) and then increase gradually if needed.

I hope this helps. All the best.

This is very helpful information Fouad, thank you very much! What about blocking? I fainly remember that in 1993 or 1994 during a brief visit in Jülich Stefan Frings in Benjamin Kaupps lab used 10 mM extracellular Magnesium to block CNG in the pinealocytes I worked with these days. The problem with inorganic substances is the potential side effects, but worth a try anyway...

I would try L-cis diltiazem, that blocks CNGA1 rod cGMP gated channels (Rispoli G, Menini A. Eur Biophys J. 1988;16(2):65-71), olfactory-type CNGA2, and cone-type CNGA3, at micromolar concentrations, applied extracellularly

People use 8-bromo-cGMP and/or *-bromo-cAMP to control gating of electrical synapses (se for example: Mills and Massey 1995). They may work with c-nucleotid gated ion channels as well.

Absolutely right Erik. Magnesium seems to be a big problem as it blocks from the intracellular side too. on this issue, see Matthews & Watanabe, 1987 and also Zimmerman and Baylor, 1991.

Giorgio Rispoli is right. Try L-cis diltiazem. Only be aware that its efficacy depends on channel assembly. The blocker is more effective on heteromeric channels than on homomeric channels. You may also activate CNG channels with 8-Br-cGMP or 8-Br-cAMP. They are membrane permeant and can be applied in the bath to activate the intracellular cyclic nucleotide binding site in the C-terminus.

Dear all, thanks for the hint to Gd3+ and L-cis-diltiazem, which I should have remembered from some papers in the late 80, early 90ies. Is there a commericial source for L-cis-diltiazem?

Also thanks for the 8-Br-cAMP suggestion, the problem is that I have PKA present and this would be activated, too. 8-Br-cGMP is worth a try.

Altogether thanks for the great suggestions!!

By the way, are there good antibodies for CNGs available, I remember both the Kaupp and the Hofmann lab had some good ones?!

I have no idea, but would specific PDE inhibitors modulate channels through affecting cAMP concentrations. One can probably buy these.

For what it's worth, many many years ago, we never got really convincing block of CNGCs with L-cis-diltizem in chicken pineal cells. Mammalian cells could be different. As already noted, its efficacy as inhibitor depends on subunit composition. I have no idea now where you might buy L-cis-diltiazem. Perhaps Tocris? Back in the day-- by which I mean about twenty years ago--it was hard to find and was not available commercially. You might be better off these days just trying to knock it down.

Gd3+ blocks the vast majority of cationic channels. It would certainly block CNGCs, but bear in mind it also blocks most TRP channels too.

Years ago I think someone also found a snake venom inibibitor.

L-cis-diltiazem is available commerically now. I recommend using a somewhat higher concentration than you would use in rods or cones.

Thanks Stuart! These are all very valuable comments!

BTW: L-cis-diltiazem is available commercially at Enzo, Santa Cruz, Abcam and problably other companies as well.

HI all,

Is that possible that myr-PKI does not reach its target, indeed the myr present on the peptide will likely sequestered it at the membrane. Alternatively you should silenced the PKACA.

Thats what I thought, too, but in another question forum on myr-PKI I opened, everyone reported good to excellent inhibition by myr-PKI-4-22. I can also transfect PKI (see Motzkus et al., 2007) or, as you suggest silence CSU, but I wanted to try a pharmacological approach this time.

Erik, do you have by chance a tag PKI expression vector for eukaryotic cells?

Indeed using 4-22 we could see a partial effect, but was much nicer with silencing the PKACA.

Sorry Daniel, the PKI construct we used had no tag. The non-tagged PKI construct was from Birgit Gellersen (Endokrinologikum, Hamburg, Germany) and if I remember correctly, she had it from Richard A. Maurer (Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, OR 97239, United States).

How did you do the PKACA-silencing? siRNA? On a plasmid? Would you be willing to provide it to us?

Dear Erik,

we used lentivirus mediated silencing (mission from sigma), we are working with mouse cell lines. maybe best is that we discuss this by email if you wish. Thanks for the tip concerning the PKI.

Best

Dear Daniel,

I agree!

My mail address is: [email protected]

Best

Erik

Hi Erik, 8-substituted PET-cGMP analogs have been shown to inhibit retinal CNG channels. If cGMP-related side effects would disturb one could either use Rp- or Sp-phosphorothioate versions, since it is only the PET modification that blocks the channel. At the moment no better tools are available, but there is an ongoing project in our lab that could result in improved structures.

Hi HG,

thanks for the reminder on the 8-PET-s! Best, Erik

Hi Erik, The best CNG antibody you can get from Alomone Labs. Their antibody(s) will work really very well. Gd3+ blocks the vast majority of cationic channels (e.g. TRPs). L-cis diltiazem blocks voltage-gated Ca2+ channels (L-, P-type as far as I remember). I used to buy it from Tocris. To activate CNG channel use db-cAMP (dibutyryl-cAMP ) and other cAMP analogs. If you find a specific blocker, I would like to know that

Dear Elena,

thanks for mentioning Alomone as the source for CNG antibodies! I ordered the CNG antisera Kit and will report here on the results when available. Thanks also for the hints regarding the "specificity" of l-cis-diltiazem, which as was and am aware of. Regarding more specific inhibitors for CNG, Stuart Dryer mentioned a snake toxin, pseudechetoxin, for which regretfully I wasn`t able to find a source for.

Very helpful for all CNG researchers is also the attached material form RBI/Sigma/Aldrich.

Finally, I cannot recommend dibutyryl-cAMP because butyrate has actions on its own, but Sp-cAMPS and Sp-cDBIMPS are potent, membrane-permenant cAMP analogs.