Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment

Vertebrate rod photoreceptors hyperpolarize when illuminated, due to the closing of cation-selective channels in the plasma membrane. The mechanism controlling the opening and closing of these channels is still unclear, however. Both 3',5'-cyclic GMP and Ca2+ ions have been proposed as intracellular messengers for coupling the light activation of the photopigment rhodopsin to channel activity and thus modulating light-sensitive conductance. We have now studied the effects of possible conductance modulators on excised 'inside-out' patches from the plasma membrane of the rod outer segment (ROS), and have found that cyclic GMP acting from the inner side of the membrane markedly increases the cationic conductance of such patches (EC50 30 microM cyclic GMP) in a reversible manner, while Ca2+ is ineffective. The cyclic GMP-induced conductance increase occurs in the absence of nucleoside triphosphates and, hence, is not mediated by protein phosphorylation, but seems rather to result from a direct action of cyclic GMP on the membrane. The effect of cyclic GMP is highly specific; cyclic AMP and 2',3'-cyclic GMP are completely ineffective when applied in millimolar concentrations. We were unable to recognize discrete current steps that might represent single-channel openings and closings modulated by cyclic GMP. Analysis of membrane current noise shows the elementary event to be 3 fA with 110 mM Na+ on both sides of the membrane at a membrane potential of -30 mV. If the initial event is assumed to be the closure of a single cyclic GMP-sensitive channel, this value corresponds to a single-channel conductance of 100 fS. It seems probable that the cyclic GMP-sensitive conductance is responsible for the generation of the rod photoresponse in vivo.     

Light-suppressible, cyclic GMP-sensitive conductance in the plasma membrane of a truncated rod outer segment

Recent experiments by Fesenko et al and ourselves have shown that excised membrane patches from retinal rod outer segments contain a cyclic GMP-sensitive conductance which has electrical properties similar to those of the light-sensitive conductance. This finding supports the notion that cGMP mediates phototransduction (see ref. 3) by directly modulating the light-sensitive conductance. However, some uncertainty remained about whether the patch experiments had discriminated completely between plasma and intracellular disk membranes; thus the cGMP response in an excised membrane could have resulted from contaminating disk membrane fragments, which are known to contain a cGMP-regulated conductance. Furthermore, the patch conductance has not yet been shown to be light-suppressible, an ultimate criterion for identity with the light-sensitive conductance. We now report experiments on a truncated rod outer segment preparation which resolved these issues. The results demonstrated that the cGMP-sensitive conductance was present in the plasma membrane of the outer segment, and that in the presence of GTP the conductance could be suppressed by a light flash. With added ATP, the effectiveness of the light flash was reduced and the suppression was more transient. The effects of both GTP and ATP were consistent with the known biochemistry. From the maximum current inducible by cGMP, we estimate that approximately 1% of the light-sensitive conductance is normally open in the dark; this would give an effective free cGMP concentration of a few micromolar in the intact outer segment in the dark.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

A cyclic GMP-activated channel in dissociated cells of the chick pineal gland.

Phototransduction in the vertebrate retina is dependent in part on a cyclic GMP-activated ionic channel in the plasma membrane of rods and cones. But other vertebrate cells are also photosensitive. Cells of the chick pineal gland have a photosensitive circadian rhythm in melatonin secretion that persists in dissociated cell culture. Exposure to light causes inhibition of melatonin secretion, and entrainment of the intrinsic circadian oscillator. Chick pinealocytes express several 'retinal' proteins, including arrestin, transducin and a protein similar to the visual pigment rhodopsin. Pinealocytes of lower vertebrates display hyperpolarizing responses to brief pulses of light. Thus it is possible that some of the mechanisms of phototransduction are similar in retinal and pineal photoreceptors. We report here the first recordings of cyclic GMP-activated channels in an extraretinal photoreceptor. Application of GMP, but not cyclic AMP, to excised inside-out patches caused activation of a 15-25 pS cationic channel. These channels may be essential for phototransduction in the chick pineal gland.     

Electrogenic Na-Ca exchange in retinal rod outer segment

Previous work has suggested that a Na-Ca exchanger may have a key role in visual transduction in retinal rods. This exchanger is thought to maintain a low internal free Ca2+ concentration in darkness and to contribute to the rod's recovery after light by removing any internally released Ca2+. Little else is known about this transport mechanism in rods. We describe here an inward membrane current recorded from single isolated rods which appears to be associated with such external Na+-dependent Ca2+ efflux activity. External Na+, but not Li+, could generate this current; high external K+ inhibited it while small amounts of La3+ (10 microM) completely abolished it. The exchanger can also transport Sr2+, but not Ba2+ or other divalent cations. The exchange ratio was estimated to be 3Na+:1Ca2+. As well as demonstrating clearly the Na-Ca exchanger in the rod outer segment, our experiments also cast serious doubt on the commonly held view that light simply releases internal Ca2+ to bind to and block the light-sensitive conductance.     

cGMP-dependent cation channel of retinal rod outer segments

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.     

Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.     

Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones

A cyclic GMP-sensitive conductance has recently been observed with patch-clamp recording in excised inside-out patches of plasma membrane from frog and toad rod outer segments. This conductance has properties suggesting that it is probably the light-sensitive conductance involved in visual transduction. We now report a similar conductance in the outer segment membrane of catfish cones. Cyclic GMP showed positive cooperativity in opening this conductance, with a Hill coefficient of 1.6-3.0 and a half-saturating cGMP concentration of 35-70 microM. Cyclic AMP at 1 mM, or changing Ca concentration (in the presence of Mg), had little effect on the conductance. In physiological solutions the cGMP-induced current had a reversal potential near +10 mV; the current amplitude increased roughly exponentially with membrane potential in both depolarizing and hyperpolarizing directions. Our results suggest that cGMP is also the internal transmitter for phototransduction in cones.     

Regulation and deregulation of cardiac Na(+)-Ca2+ exchange in giant excised sarcolemmal membrane patches

A plasmalemmal Na(+)-Ca2+ exchange mechanism is an important electrogenic determinant of contractility in cardiac cells. As in other cell types, calcium influx by Na(+)-Ca2+ exchange is secondarily activated by cytoplasmic calcium and probably ATP, but these modulatory mechanisms are either absent or altered in isolated cardiac sarcolemmal vesicles. Involvement of a calcium-dependent protein kinase in exchange regulation has been suggested but not verified. Here I describe measurements of outward Na(+)-Ca2+ exchange current, corresponding to calcium influx, in giant excised sarcolemmal patches from guinea pig myocytes. The exchange current is stimulated by both calcium and Mg-ATP from the cytoplasmic face, evidently through separate mechanisms. Activation by cytoplasmic calcium takes place within seconds, is reversible, and does not require ATP. Stimulation by Mg-ATP reverses only slowly over greater than 10 min, or not at all. Unexpectedly, a substantial decrease in exchange current occurs during activation by cytoplasmic sodium, which seems to reflect an inactivation process rather than ion concentration changes or a 'first pass' exchange cycle. This apparent inactivation, and the modulations by cytoplasmic calcium and Mg-ATP, are all abolished by brief treatment of the cytoplasmic surface with chymotrypsin, leaving the exchanger in a maintained state of high activity. Therefore, limited proteolysis deregulates Na(+)-Ca2+ exchange and could contribute to the loss of secondary regulation of the exchange in isolated sarcolemmal vesicles.     

Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel

The complete amino-acid sequence of the cyclic GMP-gated channel from bovine retinal rod photoreceptors, deduced by cloning and sequencing its complementary DNA, shows that the protein contains several putative transmembrane segments, followed by a region that is similar to the cyclic GMP-binding domains of cyclic GMP-dependent protein kinase. Expression of the complementary DNA produces cyclic GMP-gated channel activity in Xenopus oocytes.     

Structural architecture of an outer membrane channel as determined by electron crystallography.

Porins are a family of membrane channels commonly found in the outer membranes of Gram-negative bacteria where they serve as diffusional pathways for waste products, nutrients and antibiotics, and can also be receptors for bacteriophages. Porin channels have been shown in vitro to be voltage-gated. They can exhibit slight selectivities for certain solutes; for example PhoE porin has some selectivity for anionic and phosphate-containing compounds. Unlike many known membrane proteins which often contain long stretches of hydrophobic segments that are believed to traverse the membrane in a helical conformation, porins are found to have charged residues distributed almost uniformly along their primary sequences and have most of their secondary structure in a beta-sheet conformation. We have made crystalline patches of PhoE porin embedded in a lipid bilayer and have used these to determine the structure of PhoE porin by electron crystallography to a resolution of 6A. The basic structure consists of a trimer of elliptically shaped, cylindrical walls of beta sheet. Each cylinder has an inner lining, formed by parts of the polypeptide, that defines the channel size. The structure provides a clue as to how deletions of segments of polypeptide, which are found in certain mutants, can result in an actual increase in the channel size.     

Single postsynaptic channel currents in tissue cultured muscle

Some of the most compelling evidence for the existence of ionic channels in cell membranes comes from direct recording of quantised current jumps generated by the opening and closing of individual channels. Single-channel jumps have been extensively studied for lipid bilayer membranes doped with various channel-forming additives. Recently agonist-induced single-channel currents were detected in denervated frog muscle by use of extracellular electrodes, which can isolate the current from a small area of membrane. The current jumps provide a means for the direct test of many of the inferences about ionic channels which have come from electrical noise analysis. In this report we present measurements of single-channel currents induced by the agonist carbamylcholine in tissue-cultured mammalian muscle. These measurements confirm the earlier noise studies on tissue culture preparations. Recordings of single-channel currents induced by the agonist, suberyldicholine, in avian muscle are presented by Nelson and Sachs.     

副猪嗜血杆菌铁调节外膜蛋白的免疫原性研究

副猪嗜血杆菌感染已对世界养猪业造成巨大损失,作为一新发的细菌传染病,目前对其致病及免疫机理研究较少,故应用蛋白质组学及反向疫苗学技术有助于开发其新型疫苗.通过研究,鉴定出副猪嗜血杆菌的3种铁调节外膜蛋白IROMP1, IROMP B和HbpB.选择这3种蛋白进行免疫原性和攻毒保护试验研究,结果表明其中的HbpB能诱导明显的混合抗体(IgG1和IgG2a)应答,其高免血清具有明显的调理吞噬活性,HbpB能在小鼠攻毒试验中提供90%保护率,因此HbpB可成为开发副猪嗜血杆菌亚单位疫苗的候选免疫原性蛋白.