Joshua J. Singer

Direct regulation of ion channels by fatty acids

A variety of fatty acids regulate the activity of specific ion channels by mechanisms not involving the enzymatic pathways that convert arachidonic acid to oxygenated metabolites. Furthermore, these actions of fatty acids occur in patches of membrane excised from the cell and are not mediated by cellular signal transduction pathways that require soluble factors such as nucleotides and calcium. Thus, fatty acids themselves appear to regulate the action of channels directly, much as they regulate the action of several purified enzymes, and might constitute a new class of first or second messengers acting on ion channels.

Both membrane stretch and fatty acids directly activate large conductance Ca2+-activated K+ channels in vascular smooth muscle cells

Large conductance Ca2+‐activated K+ channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids. Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside‐out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca2+ from intracellular stores, or stretch induced transmembrane flux of Ca2+ or other ions potentially capable or activating the channel, Fatty acids also directly activate this channel. Like stretch activation, fatty acid activation occurs in excised inside‐out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not substrates for the cyclo‐oxygenase or lypoxygenase pathways, indicating that oxygenated metabolites do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca2+, membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.

Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction

As in many smooth muscle tissue preparations, single smooth muscle cells freshly dissociated from the stomach of the toadBufo marinus contract when stretched. Stretch-activated channels have been identified in these cells using patch-clamp techniques. In both cell-attached and excised inside-out patches, the probability of the channel being open (Po) increases when the membrane is stretched by applying negative pressure to the extracellular surface through the patch pipette. The increase inPo is mainly due to a decrease in closed time durations, but an increase in open time duration is also seen. The open-channel current-voltage relationship shows inward rectification and is not appreciably altered when K+ is substituted for Na+ as the charge-carrying cation in Ca2+-free (2 mM EGTA) pipette solutions bathing the extracellular surface of the patch. The inclusion of physiological concentrations of Ca2+ (1.8 mM) in pipette solutions (containing high concentrations of Na+ and low K+) significantly decreases the slope conductance as well as the unitary amplitude. The channel also conducts Ca2+, since inward currents were observed using pipette solutions in which Ca2+ ions were the only inorganic cations. When simulating normal physiological conditions, we find that substantial ionic current is conducted into the cell when the channel is open. These characteristics coupled with the high density of the stretch-activated channels point to a key role for them in the initiation of stretch-induced contraction.

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach ofBufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K+ selective. The probability of the channel being open (Po) increased when [Ca2+]i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca2+-activated K+ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot ofPo versus membrane potential followed the Boltzman relationship. Increasing [Ca2+]i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity ofPo residing in the mean closed time. These results were verified with a series of experiments carried out at lowPo (<0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NPo) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.

Regulation of one type of Ca2+ current in smooth muscle cells by diacylglycerol and acetylcholine.

Electrophysiological recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca2+ currents. One has a low threshold of activation and inactivates rapidly; the other has a high threshold of activation and inactivates more slowly. Acetylcholine (ACh) increased the high‐threshold current but not the low‐threshold current. The synthetic diacylglycerol analog sn‐1,2‐dioctanoylglycerol, an activator of protein kinase C (PKC), mimicked these effects of ACh on Ca2+ currents. However, another diacylglycerol analog, 1,2‐dioctanoyl‐3‐thioglycerol, which has a closely related structure but does not activate PKC, failed to increase the Ca2+ current. The same was true of l,2‐dioctanoyl‐3‐chloropropanediol, an analog that even at high concentrations only minimally activates PKC. These results suggest that diacylglycerol may be the second messenger mediating the effects of ACh on one type of voltage‐activated Ca2+ channel, possibly by activating PKC.— Vivaudou, M. B.; Clapp, L. H.; Walsh, J. V., Jr.; Singer, J. J. Regulation of one type of Ca2+ current in smooth muscle cells by diacylglycerol and acetylcholine. FASEB J. 2: 2497‐2504; 1988.

P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z‐like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of ≥300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two‐electrode voltage‐clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline+ replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO‐PRO‐12+. Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore‐forming P2Z phenotype, suggesting that ancillary factors are involved.

Visualization of Ca2+ entry through single stretch-activated cation channels

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca2+ permeable when Ca2+ is the only charge carrier, it has not been clearly demonstrated that significant Ca2+ passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca2+ fluorescence transient (SCCaFT) arising from Ca2+ entry through a single SAC opening, we provide direct evidence that significant Ca2+ can indeed pass through SACs and increase the local [Ca2+]. Results were obtained under conditions where the only source of Ca2+ was the physiological salt solution in the patch pipette containing 2 mM Ca2+. Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca2+ increase that occurred because of Ca2+ influx through voltage-gated Ca2+ channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence (“signal mass”) we determined that about 18% of the SAC current is carried by Ca2+ at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca2+ current at the resting potential. Such Ca2+ currents passing through SACs are sufficient to activate large-conductance Ca2+-activated K+ channels and, as shown previously, to trigger Ca2+ release from intracellular stores.

Cholinergic agonists suppress a potassium current in freshly dissociated smooth muscle cells of the toad.

Single micro‐electrode voltage‐clamp and current‐clamp techniques were used to study cholinergic responses in single freshly isolated gastric smooth muscle cells from the toad Bufo marinus. Acetylcholine (ACh) or muscarine caused membrane depolarization, which sometimes gave rise to action potentials and contractions. The agonist‐induced depolarization is due to the suppression of a voltage‐dependent K+ conductance, a conclusion based on the following observations. Depolarization was accompanied by an apparent membrane conductance decrease, seen as the increased size of voltage deflexions in response to constant current pulses. The conductance decrease was confirmed under voltage clamp, where current deflexions in response to constant voltage jumps were smaller in the presence of cholinergic agonists. Muscarine induced net inward currents at potentials positive to the K+ equilibrium potential (EK), and net outward currents at potentials negative to EK. In experiments where external K+ concentration ([K+]o) ranged from 20 to 90 mM the reversal potentials shifted 58 mV positive per tenfold elevation of [K+]o, as expected for a K+ current. The steady‐state current‐voltage relationship revealed that the K+ current inhibited by muscarine was larger at more positive potentials than expected from driving force considerations alone. Therefore, the underlying conductance suppressed by cholinergic agonists was voltage dependent, with almost complete deactivation at potentials more negative than approximately ‐70 mV and exhibiting a sigmoidal activation curve upon depolarization. The deactivation of this voltage‐dependent K+ conductance caused slow current relaxations to occur in response to hyperpolarizing voltage commands from depolarized holding potentials. In experiments where [K+]o ranged from 3 to 30 mM, these current relaxations reversed direction at potentials near EK and the reversal potential shifted 52 mV positive per tenfold elevation of [K+]o, indicating that K ions carry most of the charge. The current relaxations that occurred in response to hyperpolarizing voltage commands were suppressed by ACh, muscarine and oxotremorine. The effects of muscarine persisted in nominally Ca2+‐free solutions containing Mn2+. Ba2+ mimicked the effects of muscarinic agonists. Thus, isolated smooth muscle cells exhibit a K+ current resembling the M‐current of sympathetic and other neurones, which is reversibly suppressed by cholinergic agonists. The existence of a cholinergic K+ conductance decrease is of interest because it has not previously been demonstrated in smooth muscle.

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Large Conductance Ca++-Activated K+ Channels in Smooth Muscle Cell Membrane: Reduction in Unitary Currents Due to Internal Na+ Ions

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