Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes

M₂-cholinergic receptor activation by acetylcholine (ACh) is known to cause a negative inotropic and chronotropic action in atrial tissues. This effect is still controversial in ventricular tissues. The ACh-sensitive muscarinic K⁺ channel (I _K(ACh)) activity was characterized in isolated feline atrial and ventricular myocytes using the patch-clamp technique. Bath application of ACh (1 m) caused shortening of action potential duration without prior stimulation with catecholamines in atrial and ventricular myocytes. Resting membrane potential was slightly hyperpolarized in both tissues. These effects of ACh were greater in atrium than in ventricle. ACh increased whole-cell membrane current in atrial and ventricular myocytes. The current-voltage (I-V) relationship of the ACh-induced current in ventricle exhibited inward-rectification whose slope conductance was smaller than that in atrium. In single channel recording from cell-attached patches, I _K(ACh) activity was observed when ACh was induced in the pipette solution in both tissues. The channel exhibited a slope conductance of 47 ±1 pS (mean ± sd, n=14) in atrium and 47 ±2 pS (n= 10) in ventricle (not different statistically; ns). The open times were distributed according to a single exponential function with mean open lifetime of 2.0±0.3 msec (n= 14) in atrium and 1.9±0.3 msec (n=10) in ventricle (ns); these conductance and kinetic properties were similar between the two tissues. However, the relationship between the concentration of ACh and single channel activity showed a higher sensitivity to ACh in atrium (IC ₅₀ =0.03 m) than in ventricle (IC ₅₀ =0.15 m). In excised inside-out patches, ventricular I _K(ACh) required higher concentrations of GTP to activate the channel compared to atrial channels. These results suggest a reduced I _K(ACh) channel sensitivity to M₂-cholinergic receptor-linked G protein (G_i) in ventricle compared to atrium in feline heart.     

An endogenous acid-sensitive K+ channel expressed in COS-7 cells

COS-7 cells originally isolated from monkey kidney and used in many transfection studies were found to express a background K+ channel and therefore, its biophysical and pharmacological properties were examined. In cell-attached patches, a 32-pS K+ channel with a linear current-voltage relationship could be recorded. The open probability was highly voltage-dependent, with greater channel activity at depolarized potentials. The channel was markedly sensitive to changes in extracellular pH (pH(o)), showing a 70+/-10% inhibition by changing the pH(o) from 7.3 to 6.3. Arachidonic acid (5 microM) augmented channel activity 12-fold. Applying negative pressure (-40 mmHg) to the membrane patch also increased channel activity by 4-fold. These results show that COS-7 cells express a K+ channel with unique properties that must be considered when using these cells as transfection system.     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K⁺ channel responds to changes in membrane tension by undergoing a major structural change from its ‘down’ state to the more expanded ‘up’ state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K⁺ over Na⁺ during stretch activation, and that permeability to a range of other cations (Rb⁺, Cs⁺ and NH₄⁺) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K⁺ selectivity.     

Control of the cardiac muscarinic K+ channel by beta-arrestin 2

Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel.     

Physical basis for distinct basal and mechanically gated activity of the human K+ channel TRAAK

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Dominant negative effects of a Gbeta mutant on G-protein coupled inward rectifier K+ channel

HEK293 cells were transfected with cDNAs for Gbeta1(W332A) [a mutant Gbeta1], Ggamma2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gbeta1gamma2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gbeta1(W332A) suppressed this current. Most likely, Gbeta1(W332A) inhibited the action of the endogenous Gbeta. Such a dominant negative effect of Gbeta1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gbeta1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gbeta from activating the channel.     

Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.     

Intracellular K+ sensing of SKOR, a Shaker-type K+ channel from Arabidopsis

Most K+ channels in plants are structurally classified into the Shaker family named after the shaker K+ channel in Drosophila. Plant K+ channels function in many physiological processes including osmotic regulation and K+ nutrition. An outwardly rectifying K+ channel, SKOR, mediates the delivery of K+ from stelar cells to the xylem in the roots, a critical step in the long-distance distribution of K+ from roots to the upper parts of the plant. Here we report that SKOR channel activity is strictly dependent on intracellular K+ concentrations. Activation by K+ did not affect the kinetics of voltage dependence in SKOR, indicating that a voltage-independent gating mechanism underlies the K+ sensing process. Further analysis showed that the C-terminal non-transmembrane region of the SKOR protein was required for this sensing process. The intracellular K+ sensing mechanism couples SKOR activity to K+ nutrition status in the 'source cells', thereby establishing a supply-based unloading system for the regulation of K+ distribution.     

Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells

Summary A high-conductance K⁺-selective ion channel was studied in excised membrane patches from human G292 osteoblast-like osteosarcoma cells. Channel conductance averaged 170 pS in symmetric solutions of 153mm KCl, and 135 pS when the pipette was filled with standard saline (150mm NaCl). The probability of the channel being in an open state (P _open) increased with membrane potential, internal calcium, and applied negative pressure. At pCa7, channel activity was observed at membrane potentials greater than 60 mV, while at pCa3, channel activity was seen at 10 mV. Likewise, in the absence of applied pressure, channel openings were rare (P _open = 0.02), whereas with –3 cm Hg applied pressure,P _open increased to 0.40. In each case, i.e., voltage, calcium concentration, and pressure, the increase inP _open resulted from a decrease in the duration of long-closed (interburst) intervals and an increase in the duration of long-open (burst) intervals. Whole-cell responses were consistent with these findings. Hypotonic shock produced an increase in the amplitude and conductance of the outward macroscopic current and a decrease in its rise time, and both single-channel and whole-cell currents were blocked by barium. It is suggested that the voltage-gated, calcium dependent maxi-K⁺ channel in G292 osteoblastic cells is sensitive to membrane stretch and may be directly involved in osmoregulation of these cells. Further, stretch sensitivity o£ the maxi-K⁺ channel in osteotrophic cells may represent an adaptation to stresses associated with mechanical loading of mineralized tissues.     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Five-group distribution of the Shaker-like K+ channel family in higher plants

Abstract In higher plants, potassium channels of the Shaker family have been shown to play crucial roles in the uptake of K⁺ from the soil solution and subsequent transport of this ion at the cell, tissue, and organ levels. In the model plant Arabidopsis thaliana, this family is composed of nine members, which are the best characterized among plant channels at the protein, gene, and functional property levels. Plant Shaker channels share a common structure: a hydrophobic core composed of six transmembrane segments, a long cytoplasmic C-terminal region harboring a putative cyclic nucleotide binding domain, and a K_HA domain. Many channels also contain an ankyrin domain between the putative cyclic nucleotide binding domain and the K_HA domain. The analysis of 44 Shaker channels from plants revealed a five-group classification. The members of each group share high sequence and structure similarities. This grouping also correlates with the diversification of the functional properties of the proteins, as members of an individual group have roughly the same electrophysiological characteristics. Analysis of the intron positions showed that the gene structures are also quite well conserved within the five groups. A correlation linking the evolution of the sequences and the positioning of the introns was established. Finally, a moss sequence provided additional clues about the hypothetical structure of an ancestor of the present channels and suggested that the diversification of plant Shaker channels happened before the separation of monocots and dicots and after the separation of bryophytes and tracheophytes.     

Regulation by GTP of a Ca2+-activated K+ channel in the apical membrane of rabbit cortical collecting duct cells

Ca²⁺-activated K⁺ channels play an important role in Ca²⁺ signal transduction and may be regulated by mechanisms other than a direct effect of Ca²⁺. Inside-out patches of the apical membrane of confluent transformed rabbit cortical collecting duct cells cultured on collagen were subjected to patch clamp analysis. Two types of K⁺ channel, of medium and high conductance, were observed. The latter channel was characterized by a K⁺/Na⁺ permeability ratio of 10, an inwardly rectified current, a conductance of 80 pS at 0 mV, and an open probability dependent on both voltage and Ca²⁺. Guanosine 5-triphosphate (GTP) but not a guanosine 5-diphosphate (GDP) analogue, adenosine 5-triphosphate (ATP), cytidine 5-triphosphate (CTP), or inosine 5-triphosphate (ITP), inhibited the activity of this Ca²⁺-activated K⁺ channel. The inhibitory effect of GTP was dose dependent, with a 50% inhibitory concentration of 10^–5 m in the absence of Mg²⁺. In the presence of Mg²⁺ (1 mm), which is required for the binding of GTP to G proteins, the 50% inhibitory concentration decreased to 3×10^–12 m. Pertussis toxin or cholera toxin (each at 10 ng/ml) did not prevent the inhibitory effect of GTP. After removal of GTP from the medium bathing an inhibited channel, subsequent application of Ca²⁺ failed to activate the channel. Ca²⁺-activated K⁺ channels of smooth muscle cells and proximal tubule cells did not respond to GTP. Thus, the Ca²⁺-activated K⁺ channel in the apical membrane of collecting duct cells is inhibited by GTP, which appears to exert its effect via a G protein that is insensitive to both cholera and pertussis toxins.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

GORK is the only outward‐rectifying Kv‐like K⁺ channel expressed in guard cells. Its activity is tightly regulated to facilitate K⁺ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward‐rectifying K⁺ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently‐tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K⁺ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K⁺ channel blocker Ba²⁺. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo‐ and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K⁺, a characteristic that ensures the channel facilitates only K⁺ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K⁺ above 1 mm . We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K⁺.     

S641 contributes HERG K+ channel inactivation

The kinetics of voltage-dependent inactivation of the rapidly activating delayed rectifier, I _Kr, are unique among K⁺ channels. The human ether-a-gogo-related gene (HERG) encodes the pore-forming subunit of I _Kr and shares a high degree of homology with ether-a-gogo (EAG) channels that do not inactivate. Within those segments thought to contribute to the channel pore, HERG, possesses several serine residues that are not present in EAG channels. Two of these serines, S620 and S631, are known to be required for inactivation. We now show that a third serine, S641, which resides in the outer portion of the sixth transmembrane segment, is also critical for normal inactivation. As with the other serines, S641 is also involved in maintaining ion selectivity of the HERG channel and alters sensitivity to block by E4031. Larger charged or polar substitutions (S641D and S641T) disrupted C-type inactivation in HERG. Smaller aliphatic and more conservative substitutions (S641A and S641C) facilitated C-type inactivation. Our data show that, like S620 and S631, S641 is another key residue for the rapid inactivation. The altered inactivation of mutations at S620, S631, and S641 were dominant, suggesting that a network of hydroxyl side chains is required for the unique inactivation, permeation, and rectification of HERG channels.     

葛根素对豚鼠心室肌细胞钾离子通道的影响

目的 :观察葛根素对豚鼠单个心室肌细胞钾离子通道的影响。方法 :采用内面向外膜片钳单通道记录技术。结果 :葛根素 2 0 μmol/L ,4 0 μmol/L ,80 μmol/L对单个心肌细胞钾离子通道的开放概率 (P0 )有抑制作用 ,在 80μmol/L时 ,P0 值从 0 .86 7± 0 .13降至 0 .0 19± 0 .0 1,与用药前比较有显著差异 (n =5 ,P <0 .0 1)。结论 :葛根素能抑制心肌细胞钾离子通道是其抗心律失常的分子机制     

Rebuilding a macromolecular membrane complex at the atomic scale: Case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2

Ion channel‐coupled receptors (ICCR) are artificial proteins built from a G protein‐coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high‐throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2‐Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2‐Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2‐Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ‐opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2‐Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N‐terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2‐Kir6.2 construct. Proteins 2014; 82:1694–1707. © 2014 Wiley Periodicals, Inc.