Quantifying the cooperative subunit action in a multimeric membrane receptor

Self Cite

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.

Section: Discussionsupporting

confidence: 83%

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation

Otte

Schweinitz

Bonus

et al. 2018

Self Cite

“…3b ). Notably, both these apparent affinities are higher than that of one CNGA2 subunit (EC 50 = 22.2 μM cGMP) in concatameric homotetrameric CNGA2 channels with three disabled subunits 30 . It will have to be shown by future experiments whether this value holds also for a single CNGA2 subunit in heterotetrameric CNGA2:A4:B1b channels.…”

Section: Discussionmentioning

confidence: 92%

Deciphering the function of the CNGB1b subunit in olfactory CNG channels

Nache

Wongsamitkul

Kusch

et al. 2016

Self Cite

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.Cyclic nucleotide-gated (CNG) channels are nonselective cation channels that play an important role for the sensory signaling in the vertebrate visual and olfactory system 1 . These channels are activated by the binding of cyclic nucleotides (cAMP or cGMP) 2,3 . Native CNG channels are heterotetramers composed of two or three different types of subunits: 3 x CNGA1 and 1 x CNGB1a in rod channels 4,5 , 2 x CNGA3 and 2 x CNGB3 in cone channels 6 , 2 x CNGA2, 1 x CNGA4 and 1 x CNGB1b in olfactory channels 7,8 . All subunits share a similar architecture, containing six transmembrane domains (S1 to S6), a pore region between S5 and S6 and an intracellulary located Nand C-terminus. At the C-terminus each subunit contains a cyclic nucleotide-binding domain (CNBD) 2 . Despite these structural similarities, only CNGA1, CNGA2 and CNGA3 subunits are able to form functional channels on their own when expressed heterologously, whereas the CNGB and CNGA4 subunits do not [9][10][11] . These subunits were therefore considered to be modulatory, and knowledge about the type of modulation has been growing over the last years.For the heterotetrameric olfactory CNG channels it is known for long that the presence of both the CNGB1b and CNGA4 subunit leads to an increased apparent affinity for cyclic nucleotides 7,8,10,12,13 and accounts for the fast inhibition of native channels by Ca 2+ /CaM 14-16 . In particular the CNGB1b subunit was shown to be critical for Ca 2+ /CaM-mediated desensitization 17 . Moreover, the CNGB1b subunit is also responsible for the ciliary trafficking of the native heterotetrameric channel 18 . Although all three types of subunits in olfactory CNG channels have a binding domain, it is still elusive whether or not all of them indeed bind a ligand in the heterotetrameric context and, if so, to what extent they actively contribute to channel activation. Studying ligand binding to...

“…Major progress in understanding the function of P2X receptors can be expected from applying global fit analyses of extended data, as demonstrated previously for other ion channels. Using current data at equilibrium this has been demonstrated for concentration-activation relationships of concatenated CNGA2 channels, containing a different number of disabled binding sites 19 , for IV -relationships of viral Kv channels at different concentrations of permeating K + ions 20 , or gating charge movement in Shaker channels 21 . We previously also globally fitted data distant from equilibrium by kinetic schemes, for activation time courses of tetrameric CNGA2 channels induced by cGMP jumps to different concentrations 22 and voltage-induced activation time courses of tetrameric HCN2 channels 23 .…”

Section: Introductionmentioning

confidence: 99%

Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels

Sattler

Eick

Hummert

et al. 2020

Self Cite

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.