Asymmetric Interplay Between K+ and Blocker and Atomistic Parameters From Physiological Experiments Quantify K+ Channel Blocker Release

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels.

Effects of ionic strength on gating and permeation of TREK-2 K2P channels

In addition to the classical voltage-dependent behavior mediated by the voltage-sensing-domains (VSD) of ion channels, a growing number of voltage-dependent gating behaviors are being described in channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel, TREK2 responds to membrane voltage through a gating process mediated by the interaction of K+ with its selectivity filter. Recently, we found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found TREK-2 is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is complex.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels

In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+ with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.

Unraveling of a Strongly Correlated Dynamical Network of Residues Controlling the Permeation of Potassium in KcsA Ion Channel

The complicated patterns of the single-channel currents in potassium ion channel KcsA are governed by the structural variability of the selectivity filter. A comparative analysis of the dynamics of the wild type KcsA channel and several of its mutants showing different conducting patterns was performed. A strongly correlated dynamical network of interacting residues is found to play a key role in regulating the state of the wild type channel. The network is centered on the aspartate D80 which plays the role of a hub by strong interacting via hydrogen bonds with residues E71, R64, R89, and W67. Residue D80 also affects the selectivity filter via its backbones. This network further compromises ions and water molecules located inside the channel that results in the mutual influence: the permeation depends on the configuration of residues in the network, and the dynamics of network’s residues depends on locations of ions and water molecules inside the selectivity filter. Some features of the network provide a further understanding of experimental results describing the KcsA activity. In particular, the necessity of anionic lipids to be present for functioning the channel is explained by the interaction between the lipids and the arginine residues R64 and R89 that prevents destabilizing the structure of the selectivity filter.

NMDA receptor channel gating control by the pre-M1 helix

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Ion channels and disease

Ion channels are membrane proteins that act as gated pathways for the movement of ions across cell membranes. They are found in both surface and intracellular membranes and play essential roles in the physiology of all cell types. An ever-increasing number of human diseases are now known to be caused by defects in ion channel function. To understand how ion channel defects give rise to disease, it is helpful to understand how the ion channel proteins work. This chapter therefore considers what is known of ion channel structure, explains the properties of the single ion channel, and shows how single-channel currents give rise to action potentials and synaptic potentials.

IKs ion-channel pore conductance can result from individual voltage sensor movements

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

Nitric oxide augments single persistent Na+ channel currents via the cGMP/PKG signaling pathway in Kenyon cells isolated from cricket mushroom bodies

The nitric oxide (NO)/cyclic GMP signaling pathway has been suggested to be important in the formation of olfactory memory in insects. However, the molecular targets of the NO signaling cascade in the central neurons associated with olfactory learning and memory have not been fully analyzed. In this study, we investigated the effects of NO donors on single voltage-dependent Na+ channels in intrinsic neurons, called Kenyon cells, in the mushroom bodies in the brain of the cricket. Step depolarization on cell-attached patch membranes induces single-channel currents with fast-activating and -inactivating brief openings at the beginning of the voltage steps followed by more persistently recurring brief openings all along the 150-ms pulses. Application of the NO donor S-nitrosoglutathione (GSNO) increased the number of channel openings of both types of single Na+ channels. This excitatory effect of GSNO on the activity of these Na+ channels was diminished by KT5823, an inhibitor of protein kinase G (PKG), indicating an involvement of PKG in the downstream pathway of NO. Application of KT5823 alone decreased the activity of the persistent Na+ channels without significant effects on the fast-inactivating Na+ channels. The membrane-permeable cGMP analog 8Br-cGMP increased the number of channel openings of both types of single Na+ channels, similar to the action of NO. Taken together, these results indicate that NO acts as a critical modulator of both fast-inactivating and persistent Na+ channels and that persistent Na+ channels are constantly upregulated by the endogenous cGMP/PKG signaling cascade. NEW & NOTEWORTHY This study clarified that nitric oxide (NO) increases the activity of both fast-inactivating and persistent Na+ channels via the cGMP/PKG signaling cascade in cricket Kenyon cells. The persistent Na+ channels are also found to be upregulated constantly by endogenous cGMP/PKG signaling. On the basis of the present results and the results of previous studies, we propose a hypothetical model explaining NO production and NO-dependent memory formation in cricket large Kenyon cells.