Vertebrate Smooth Muscle Research Articles

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 m m) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 m m) it potentiates the ATPase activity three-to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin—actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca 2+-sensitive reversible bundling effect. Comparison of our data with those of contracting smooth muscle suggests that bundling of thin filaments may be important in smooth muscle contraction.

Smooth muscle myosin is composed of homodimeric heavy chains.

Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115 microns/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078 microns/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082 microns/s).

Calponin and the composition of smooth muscle thin filaments

Recent work suggests that caldesmon and calponin, two actin-binding proteins found in smooth muscle, compete with each other for interaction with F-actin, implying that they may not be subunits of the same complex in vivo. Rather than associating with the same thin filaments in vivo, the two proteins actually may be localized on separate filaments possibly performing distinct functions. It is well-established that actomyosin ATPase and consequently tension in vertebrate smooth muscle are dependent on Ca2+-calmodulin-activated phosphorylation of myosin light chain subunits (reviewed in Kamm & Stull, 1985). The possibility of additional thin filament-linked modulation, raised by the pioneering work of Driska and Hartshome (1975) and Ebashi and colleagues (1977) and detailed extensively by Marston and colleagues (Marston et al., 1980; Marston & Smith, 1984), also has received much attention. This possibility became particularly attractive after Sobue and colleagues (1981) first isolated and characterized the protein caldesmon, which now is known to be localized exclusively on thin filaments (Marston & Lehman, 1985) and capable of modulating actomyosin ATPase in vitro (Sobue eta]., 1981; Ngai & Walsh, I984; Dabrowska et al., 1985; Marston & Lehman, I985; Chacko et al., I987). More recently, a second putative thin filament-linked regulatory protein, calponin, has been characterized by Takahashi and colleagues (1986, 1988, 1990), and their work on this 34-35 kDa molecule has stimulated further interest in the role played by thin filament-binding proteins in the contractility of smooth muscle. In these studies, antibody crossreactivity and molecular cloning demonstrated that regions of calponin have an amino acid sequence homologous to one of the tropomyosin binding domains of skeletal muscle troponinT, suggesting strongly that calponin is a true thin filament binding protein (Takahashi et al., 1988, 1990). Calponin also shares extensive sequence homology with the very abundant and modestly-named smooth muscle protein, SM 22, which as yet has no known function or binding affinities (Vancompernolle et al., 1990; Takahashi et al., 1990; Lees-Miller et al., 1987; Pearlstone et al., 1987). Calponin, like caldesmon, binds to actin with high affinity and also binds calmodulin (Lehman & Kaminer, 1984; Takahashi et al., 1986; Lehman, 1989; Winder & Walsh, 1990; Sutherland eta]., I990). However, with both caldesmon and calponin potentially interacting with actin, models of smooth muscle thin filaments seem to be becoming somewhat congested. Clearly a more complete understanding of the composition, assembly, and distribution of thin filaments in smooth muscle is required to clarify the roles played by these thin filament-binding proteins. A number of studies analyzing reconstituted and intact thin filaments have begun to address these questions and provide direction for future investigation. Experiments in the laboratories of Shirinsky and Dabrowska may have yielded a key piece of evidence pertinent to our understanding of how caldesmon and calponin interact with actin. In their studies, F-actin complexed with purified calponin was exposed to increasing concentrations of caldesmon; this resulted in binding of the caldesmon and displacement of bound calponin. Analogously, they also found that calponin was able to displace caldesmon bound to actin filaments (Makuch eta]., 1991). Hence, caldesmon and calponin compete for actin binding sites on thin filaments and apparently do not form a complex with each other, as do the subunits of troponin on skeletal muscle thin filaments. This would explain why, in some preparations, calponin seems to interfere with caldesmon inhibition of actomyosin ATPase (S. B. Marston, personal communication) and, in others, caldesmon with calponin inhibition (Sutherland eta]., 1990). Surprisingly, the presence of tropomyosin does not seem to influence calponin inhibition of ATPase (Makuch eta]., 1990; Winder & Walsh, 1990), and its role in calponin function and in calponin binding on thin filaments remains undefined. Even though calponin and caldesmon both bind to actin, it seems unlikely that they are randomly or even sequentially arranged on individual thin filaments in vivo. Caldesmon, at least, is regularly and periodically distributed on relatively intact, isolated, thin filaments in

Phosphorylatioh of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells

In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate non-muscle myosin heavy chains both in vitro and in intact cells. A single serine residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single serine residue located near the carboxyl terminus of the 204 x 10(3) Mr smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphorylate the 200 x 10(3) Mr smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204 x 10(3) Mr myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.

Supercontracted state of vertebrate smooth muscle cell fragments reveals myofilament lengths.

Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.

Calcium regulated thin filaments from molluscan catch muscles contain a caldesmon-like regulatory protein

The thin filaments of the anterior byssus retractor muscle of the edible mussel Mytilus and the transluscent and opaque adductors of the oyster Crassostrea have been isolated and their properties investigated. We find that the thin filaments from all three muscles can activate skeletal muscle myosin ATPase in the presence of calcium but that the activity is inhibited in its absence. The filaments contain a protein which interacts with antibodies to vertebrate smooth muscle caldesmon on immunoblots. The antibodies relieve the inhibition of the thin-filament-activated myosin MgATPase. They can also bundle the thin filaments. We conclude that a caldesmon-like protein is present in molluscan muscle. As in the vertebrate smooth muscle, it could act as part of a control mechanism in addition to the myosin regulatory system. Vertebrate smooth muscle caldesmon can crosslink actin and myosin and it has been suggested that it may in this way contribute to the latch state. A similar interaction may be involved in the catch mechanism in molluscan muscle.

Caldesmon and the Structure of Vertebrate Smooth Muscle Thin Filaments

Caldesmon and the Structure of Vertebrate Smooth Muscle Thin Filaments

Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments

Native and synthetic vertebrate smooth muscle thin filaments have been examined by electron microscopy in order to determine the arrangement of the regulatory protein caldesmon. In synthetic filaments of actin-caldesmon, long slender molecules were sometimes seen running along the thin filament, suggesting that caldesmon can associate with actin along its length, while at other times lateral projections were observed. In native filaments, containing actin, caldesmon and tropomyosin, we found no evidence for lateral projections extending from the filaments, suggesting that caldesmon does not act as a crosslinking protein in vivo. In contrast, elongated molecules were clearly seen following the long pitch actin helices. We suggest that these may represent an association of caldesmon and tropomyosin. Antibodies developed against an N-terminal fragment of caldesmon caused thin filaments to aggregate laterally into arrays displaying approximately 35-38 nm repeats; thin filament aggregates with this periodicity were obtained previously (Lehman et al., 1989) using antibodies to the C-terminal segment of caldesmon. These results suggest that both ends of caldesmon are closely associated with the shaft of the thin filament, supporting a model in which the elongated caldesmon molecule runs along the filament, possibly interacting with tropomyosin, following the long pitch actin helices.

The retrieval and display of patent images from optical storage

The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a λgt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5′ end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O. volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40–60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.

35 kDa proteins are not components of vertebrate smooth muscle thin filaments.

A 35 kDa protein present in vertebrate smooth muscle and capable of binding to purified actin does not appear to be a constituent of smooth-muscle thin filaments in vivo; instead, it is more likely to be a component easily solubilized from particulate material which then spuriously interacts with actin.

Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar

The structure of myosin filaments isolated from skinned toad stomach smooth muscle cells has been examined by electron microscopy as a step toward identifying the in vivo structure. When negatively stained following exposure to relaxing conditions, the filaments exhibited a continuous 14-nm axial repeat of crossbridge projections with no central bare zone. The filaments thus differed from the bipolar filaments found in striated muscle and displayed instead features resembling side-polar and mixed-polarity filament models. By rotation of isolated filaments around their longitudinal axes it was found that cross bridges occurred only along two sides of the filament, an arrangement consistent with the side-polar but not the mixed-polarity model. The polarity is thus similar to that proposed for ribbons (Small & Squire, J. molec. Biol. 67, (1972) 17-149) and for synthetic smooth muscle myosin filaments (Craig and Megerman, J. Cell Biol. 75, (1977) 990-996); their appearance in cross-section, however, shows that these structures are filaments (i.e. with two axes of similar dimensions) and not broad ribbons. As the filaments were derived directly from skinned cells which contracted and relaxed in response to physiological levels of MgATP and Ca2+ at rates comparable to those of native, isolated cells, this unusual arrangement of cross bridges appears to be an effective, functional form of myosin in the contractile apparatus. Side-polar filaments therefore merit consideration as plausible candidates for the native organization of myosin in vertebrate smooth muscle cells.

Kinins in ant venoms—A comparison with venoms of related hymenoptera

1. Venom preparations have been made of six ant, one pompilid wasp, two mutillid wasp, and four social wasp species. 2. The venoms were analysed pharmacologically in order to detect kinin-like activity. 3. Due to the small amounts of venoms available only a cascade of smooth muscle preparation could be used. 4. Kinin activities have been found in five ant venoms and in four social wasp venoms. 5. No kinin activity has been found in the venoms of the pompilid and mutillid wasps. 6. All ant venoms also contain unidentified agonists for vertebrate smooth muscle preparations.

Bulk isolation of the 20,000-Da light chain of smooth muscle myosin: Separation of the unphosphorylated and phosphorylated species

The procedure of W. T. Perrie and S. V. Perry (1970, Biochem. J. 119, 31–38) has been improved and extended to allow a convenient large-scale isolation of the 20,000-Da light chain of vertebrate smooth muscle myosin. The method utilizes as source material tropomyosin-free actomyosin or myosin. The relatively pure light chain isolated from this material could be obtained in pure form by a single gel-filtration step. Separation of the unphosphorylated and phosphorylated light chain species was achieved by subsequent chromatography on a DEAE column. The solubility properties of this light chain, relevant to its use in myosin light chain kinase assays, were also established.

Muscle Cells of Hollow Organs

Muscle cells in hollow organs must shorten and perform work much like skeletal muscle. However, they must also contract tonically to maintain organ dimensions against imposed loads. Vertebrate smooth muscle has a regulatory system involving Ca2+-stimulated cross-bridge phosphorylation that controls not only the number of cross bridges interacting with the thin filament but also controls their cycling rates. Regulation by phosphorylation and dephosphorylation lowers the efficiency of smooth muscle but contributes to a remarkable economy of force maintenance.

Ionic currents of smooth muscle cells isolated from the ctenophoreMnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.

Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig protal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an approximately 45 degree angle. In the absence of Ca2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some cross-bridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM Pi, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca2+, was estimated to be 0.1-2.5 X 10(5) M-1s-1, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca2+, force development occurred at a rate (0.4 s-1) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP gamma S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca2+, consistent with cooperative attachment of cross-bridges. Cooperative reattachment of dephosphorylated cross-bridges may contribute to force maintenance at low energy cost and low cross-bridge cycling rates in smooth muscle.

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence: Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) hase been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated M r of 229 × 10 3, and can be divided into a long α-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two “hinges” for the 6S/10S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg 2+ ATP is bound to a pocket between two opposing α β domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Identification and characterization of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique

Voltage-clamp experiments were carried out on freshly dissociated single vertebrate smooth muscle cells from the stomach muscularis of Bufo marinus. Conventional two-microelectrode methodology was used, thus avoiding rapid dialysis of the cytosol. Four major phases of current were identified upon voltage jumps from negative holding levels to more positive levels. The first phase of current was an initial, inward current. This current was blocked by external Mn2+ and was of the correct magnitude to account for the rising phase of the Ca2+-dependent, TTX-independent action potentials found in these cells. Following this initial, inward Ca2+ current, a large outward current was observed which reached its peak over a period of hundreds of milliseconds and then decayed over a period of seconds to a steady-state level. The peak outward current and the steady-state outward current constitute the second and third major currents. The peak outward current was the largest current observed, with a magnitude as large as tens of nanoamps whereas the inward current was at most about one nanoamp. The peak outward current was reduced more than tenfold in the presence of external TEA. It was also decreased or abolished when the preceding inward current was diminished or eliminated by using external Mn2+ or less negative holding potentials. In this way the peak outward current was identified as a Ca2+-activated K+ current whose slow decay was hypothesized to result from removal of internal Ca ions by cellular mechanisms following the initial rise in [Ca2+]i resulting from the inward current. A fourth major current was an early transient outward current observed most clearly upon voltage jumps to more positive potentials when the inward current was eliminated by using less negative holding potentials or external Mn2+. A classical steady-state inactivation relationship as a function of membrane potential was constructed for the inward current. A substantial portion of this inactivation curve lies at potentials negative to the apparent threshold for activation of inward current, suggesting a true voltage-dependent inactivation. Although additional Ca2+-dependent inactivation could not be ruled out, neither could evidence for it be found.

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 of Bufo 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 of Po versus membrane potential followed the Boltzmann 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 of Po residing in the mean closed time. These results were verified with a series of experiments carried out at low Po (less than 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.