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.     

On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.     

Indirect activation of the epithelial Na+ channel by trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.     

Determining K+ channel activation curves from K+ channel currents

Potassium ion channels are generally believed to have current-voltage (IV) relations which are linearly related to driving force ( V - E(K)), where V is membrane potential and E(K) is the potassium ion equilibrium potential. Consequently, activation curves for K+ channels have often been measured by normalizing voltage-clamp families of macroscopic K+ currents with (V - E(K)), where V is the potential of each successive step in the voltage clamp sequence. However, the IV relation for many types of K+ channels actually has a non-linear dependence upon driving force which is well described by the Goldman-Hodgkin-Katz relation. When the GHK dependence on (V - E(K)) is used in the normalization procedure, a very different voltage dependence of the activation curve is obtained which may more accurately reflect this feature of channel gating. Novel insights into the voltage dependence of the rapidly inactivating I(A) channels Kv1.4 and Kv4.2 have been obtained when this procedure was applied to recently published results.     

Molecular identification of the Na+-activated K+ channel

Progress in understanding sodium-activated potassium channels (K(Na)), suggested to function in excitable cells both during physiological conditions and protectively during hypoxia, has been limited by their unknown molecular identity. In this issue of Neuron, Salkoff and coworkers now show that members of the Slo gene family, Slo2.1 and Slo2.2, encode functional K(Na) channels.     

Membrane stretch augments the cardiac muscarinic K+ channel activity

Arachidonic acid has been shown to activate K⁺-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (G _K )-gated, muscarinic K⁺ (K_ACh) channel can also be activated by arachidonic acid, we investigated whether the K_ACh channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), K_ACh channels were not active, and negative pressure failed to activate these channels. With ACh (10 m) in the pipette, applying negative pressure (0 to –80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and inside-out patches (100 m GTP in bath). Membrane stretch did not alter the sensitivity of the K_ACh channel to GTP. When G _K was maximally activated with 100 m GTPS in inside-out patches, the K_ACh channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the K_ACh channel in the absence of ACh and GTP; K_ACh channel activity was further increased by stretch. These results indicate that the atrial muscarinic K⁺ channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.     

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

MthK is a Ca²⁺-gated K⁺ channel whose activity is inhibited by cytoplasmic H⁺. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H⁺ inhibition and Ca²⁺-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca²⁺] (ranging from nominally 0 to 30 mM) at a given [H⁺], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca²⁺] and open probability (Po), with a mean Hill coefficient (n_H) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor n_H changed significantly as a function of [H⁺] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca²⁺ was not H⁺ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H⁺] raised the EC₅₀ for Ca²⁺ activation by ∼4.7-fold per tenfold increase in [H⁺], displaying a linear relation between log(EC₅₀) and log([H⁺]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H⁺ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca²⁺ binding and channel opening. We can account for the Ca²⁺ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca²⁺ allosterically activates MthK, whereas H⁺ opposes activation by destabilizing the binding of Ca²⁺.     

Na+ block and permeation in a K+ channel of known structure

The effects of intracellular Na(+) were studied on K(+) and Rb(+) currents through single KcsA channels. At low voltage, Na(+) produces voltage-dependent block, which becomes relieved at high voltage by a "punchthrough" mechanism representing Na(+) escaping from its blocking site through the selectivity filter. The Na(+) blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K(+) and Rb(+) against Na(+). The voltage dependence of Na(+) block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Toxin-induced K+ efflux through the Na+ channel of neuroblastoma cells

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+ influx through this channel also give rise to the efflux of 86Rb+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42K+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

Na+, K+ -ATPase: causes of activation of K+ -phosphatase reaction by nucleotides

Hydrolysis of umbelliferone phosphate is inhibited by sodium ions and ATP added to the incubation medium together with Na+ activates it, CTP, ITP, UTP and GTP produce the same effect. Acetyl phosphate, p-nitrophenyl phosphate, ADP and adenylyl imidodiphosphate beta, gamma-NH-ATP, ATP nonhydrolyzable analog, have no activating effect. ATP produces a mixed inhibition of umbelliferone phosphate hydrolysis both in the presence and absence of Na+. A scheme is suggested which explains the mechanism of the nucleotide activating effect.     

Stimulation of Na+-dependent amino acid uptake by activation of the Ca2+-dependent K+ channel in the Ehrlich ascites tumor cell

The activation of Ca²⁺-dependent K⁺ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na⁺-dependent transport of α-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K⁺ conductance. The same treatment does not modify the Na⁺-independent uptake of the norbornane amino acid.     

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.     

Stimulation of Na+ -dependent amino acid uptake by activation of the Ca2+ -dependent K+ channel in the Ehrlich ascites tumor cell

The activation of Ca2+ -dependent K+ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na+ -dependent transport of alpha-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K+ conductance. The same treatment does not modify the Na+ -independent uptake of the norbornane amino acid.     

C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism.

The lymphocyte voltage-gated K+ channel, Kv1.3, inactivates by a C-type process. We have elucidated the molecular basis for this process using a kinetic analysis of wild-type and mutant (A413V) Kv1.3 homo- and heteromultimeric currents in a mammalian lymphoid expression system. The medians of the measured inactivation time constants for wild-type and A413V homotetrameric currents are 204 and 4 ms, respectively. Co-expression of these subunits produces heteromultimeric channels manifesting inactivation kinetics intermediate between those of wild-type and A413V homomultimers. We have considered several models in which each subunit acts either independently or cooperatively to produce the observed inactivation kinetics. The cooperative model gives excellent fits to the data for any heteromultimeric composition of subunits, clearly distinguishing it from the independent models. In the cooperative model, the difference in free energy between the open and inactivated states of the channel is invariant with subunit composition and equals approximately 1.5 kcal/mol. Each subunit contributes equally to the activation free energy for transitions between open and inactivated states, with an A413V subunit decreasing the free energy barrier for inactivation (and for recovery from inactivation) by approximately 0.6 kcal/mol. Our results are consistent with a physical model in which the outer mouth of the channel constricts during C-type inactivation (G. Yellen, D. Sodickson, T. Chen, and M.E. Jurman, 1994, Biophys. J. 66:1068-1075).