1.The In Vitro Effects And Mechanism Of Low Power LaserIrradiation On Hemortheological Parameters 2.Intersubunit Interaction Within The Cytoplasmic Domain Of Inwardly Rectifying Potassium Channels Is A Structural Determinant For Channel Activation

PART IThe ability of laser irradiation to destroy tissue is well known. Less known is the fact that the same radiation, at much lower intensities, can non-destructively alter cellular function. This latter phenomenon, which is called low power laser "biostimulation" effect, is now a basis for the conservative treatment of a variety of diseases including cardiac and vascular conditions, neurological conditions, wound healing, bactericidal effects, dental surgery et al.The intravascular irradiation of low power laser has been applied in pre-clinical and clinical to treat various pathological processes. However, the mechanism is not fully understood so far. Especially, the interaction and related mechanism between the laser and blood are kept unclear. In this work, by measuring the changing of some main rheological factors after laser irradiation, the interaction and mechanism were explored.A 30 mW He-Ne laser was used for irradiation with the 4 mm diameter of beam spot on blood samples. The pathological samples of blood were obtained from patients (volunteers), and each sample was divided into 2 tubes for irradiation and control. The blood viscosity, erythrocyte deformability and sedimentation rate were measured after laser irradiation and compared with un-irradiated control. The blood samples with poor erythrocyte deformability were prepared by adding the Ca²⁺ to the normal erythrocytes Results showed that laser irradiation reduced the erythrocyte sedimentation rate of blood samples, which had hyper-sedimentation rate originally. The blood viscosity of samples in hyper-values was lowered by laser irradiation in all shear rates measured (10 - 110 S^-1) with the relative variation around 10%. The deformability of erythrocytes from pathological samples and Ca²⁺ treated samples was all improved after laser irradiation. The final conclusion is in vitro He-Ne laser irradiation has the positive effects on improving the rheological properties of blood.Using the blood samples of animal, the in vitro effects of low power laser irradiation on erythrocyte rheology were investigated. After the deposited pig's erythrocytes (the deformation of erythrocytes have already turned toworse) were irradiated with laser, the effects of laser irradiation on erythrocyte' s deformation were measured. When 650 nm laser was used for irradiation, the deformability of erythrocytes was improved obviously. The changing tendency is that, the erythrocyte' s deformation was enhanced with the increasing of irradiation power and then saturated around 4⁵ mW. When the blood samples were irradiated by 650 nm and 632. 8 nm lasers respectively with the same power (10 mW), the erythrocyte deformability in two cases were all obviously increased to the similar level. These phenomena can be explained by that, the hemoglobin in erythrocytes has the similar absorption to both lasers. Taking the mouse blood as the samples, the effects of laser irradiation on erthrocyte' s electrophoretic mobility was studied further. When irradiated by 632.8 nm laser, the electrophoretic mobility of erythrocytes was speeded also, reflecting the electric charges on the surface of erythrocytes were increased which will help to decrease the aggregation of erythrocytes. Under such low power irradiation (less than 20 mW), no morphological change was detected by the microscopy and no hemolysis was found also.The effects of laser irradiation with 632.8 and 532 nm on rheological properties of blood were comparatively studied in vitro. Under the irradiation condition of 30 raw, laser irradiation of blood samples using a spot diameter of 5 mm with each laser, showed promising results in the modulation of hemorheological properties. When blood samples from patients with abnormally high values of erythrocyte sedimentation rate (ESR) were Irradiated, the values of ESR were lowered statistically by either of the 632.8 or 532 nm lasers. The laser irradiation reduced blood viscosities at different shear rates (10 110 S"1) for the hyper-viscosity blood samples. The erythrocyte deformability was enhanced by laser irradiation when the deformability of the sample from the patients was originally poor. For further explore the improvement of laser irradiation on erythrocyte deformability, the typical erythrocyte samples with poor deformability were produced by the pre-treatment of the erythrocytes with Ca2*. The deformabili ty of these Erythrocyte samples was also improved after laser irradiation.As we known, the main content in erythrocyte is hemoglobin, and it was reported and be confirmed that the membrane attached hemoglobin (Hbm) is thedeterminant factor of erythrocyte deformability. The more Hbm molecules an erythrocyte contains, the worse its deformability is. The Hbm has broad absorption band in visible region, so the Hbm might be the target molecules in laser irradiation.When erythrocytes were treated with CaCl2 or ionophore A23187, the Hbm in these treated erythrocytes increased obviously and the erythrocyte deformability was turned to worse accordingly. These treated erythrocytes were irradiated with 632. 8 and 532 nm laser respectively, and then the content changing of Hbm was measured. The results show that both 532 nm (30 mW, 20 minutes) and 632.8 nm (30 mw, 60 minutes) irradiation lower the Hbrn as well as increase the deformability of erythrocytes, indicating that the Hbm is the real target molecule under laser irradiation.In all experiments including ESR, blood viscosity, EPM and erythrocyte deformability, the 532 nm laser demonstrated more efficient effects on modulating rkeological properties than 632.8 nm laser. This wavelength effect is consistent with the absorption spectrum of hemoglobin, reflecting that hemoglobin may be one of the action targets under laser irradiation.PART IIIon channels are membrane protein complexes and their function is to facilitate the diffusion of ions across biological membranes.K⁺ channels specifically allow potassium ions to cross the cell membrane through a water-filled channel protein. Open K channels stabilize the membrane potential: they draw the membrane potential closer to the potassium equilibrium potential and farther from the firing threshold. Inwardly rectifying potassium channels (Kir) is a term which describes the fact that potassium channels through which the inward flow of K⁺ ions into the cell is greater than the outward flow. Kir channels set and regulate the resting membrane potential of the cell and thereby modulate the electrical activity of cardiac and neuronal cells, insulin secretion, and epithelial K⁺ transport. Kir is a big family, including seven major subfami lies. Kir channels are much smaller than voltage-gated K⁺ (Kv) channels, possessing only two putative transmembrane domains, TM1 and TM2, linked by a loop which dips back down into the membrane to line the outer part of the pore. This loop is refored to as the pore loop (or H5 region) and it shows significant sequence homology to the pore loops of other K⁺ channels. A characteristic of inward rectifiers is that they are subject to modulation by a variety of cytoplasmic agents. Of particular physiological importance is the regulation produced by protons, phosphorylation, GTP^binding proteins and adenine nucleotidesATP-sensi tive K⁺ channels (KATP channels), first discovered in the heart, link the membrane potential to the metabolic state of the cell as reflected by the levels of nucleoside triphosphates and diphosphates. But they have now been identified in most excitable cells including pancreatic β-cells, neurons, cardiac myocytes, skeletal and smooth muscle cells. KATP channels are major drug targets in pancreatic, vascular smooth muscle and cardiac muscle. Most types of KATP channel are blocked by sulphonylurea(磺脲) drugs, such as glibenclamide and tolbutamide, which are used in the treatment of non-dependent diabetes, and they are activated by KH channel openers, such as diazoxide(氯甲苯噻嗪) and cromakalim. As a result of recent progress in molecular biology and electrophysiology, KATP channel structure anddiversity is increasingly being understood. Cloning, sequencing, and functional analyses of KATP channel genes reveal different molecular composition of the various KATP channels. It is now well known that plasma membrane KATP channels consist of two different structural subunits: an inwardly rectifying potassium channel subunit, which forms the pore ( Kir6. x ), and a sulfonylurea receptor (SURX) as the regulatory subunit. The subunits assemble as a hetero-octamer with 4:4 stoichiometryKATP is not only a physiological important molecular but also subject to very sophisticated regulation. KATP channels can be regulated through various pathways inside the cellular environment.The major regulations are nucleotide. Other regulators include PPIs, phosphorylation, G-proteins, PH and a number of pharmacological reagents.Mutation-functional analysis has been revealed many details of this channel. The structural basis of KATP function was approached mainly using point-mutations, chimeric switches and cysteine screening combined with covalent modification using membrane-impermeable methanethiosulfonats ( MTS reagents). It became clear that primary ATP binding site responsible for ATP inhibition is located on Kir6. 2 subunit, after Tucker et al showed that Kir6. 2AC26, a C-terminal truncated Kir6. 2 that can express without SUR, actually forms an ATP-inhibitable channel. Thus we have focused on Kir6. 2 to search for the mechanism of channel gating and related modulators such as PPIs and PH.Although many reports support a model in which ATP, phosphoinositides and hydrogen ion bind directly to the cytoplasmic domains of Kir6. 2 to promote channel closure and opening, respectively, little is known about the tertiary or quaternary structural elements in these domains that are involved in ligand binding and channel gating.There are two gating kinetics of the Kir channels. One is intrinsic gating which has faster kinetics, insensitive to regulation. The other is controlled gating which has slower kinetics, controlled and regulated by modulators, determines the physiological functions of Kir channels in cellsPrevious studies have shown that the controllable gate is located at the helix-bundle crossing and most Kir channel modulators interact with the cytoplasmic domain. Since the controlled gating of Kir channels isaccomplished by displacement of the cytoplasmic domain, a process in which subunit-subunit interaction must be involved. The specific hypothesis we will test is intersubunit interaction within the cytoplasmic domain is critical to the channel gating.Structural studies have suggested that displacement of the cytoplasraicsegment is likely to induce movement of the crossed bundle that presumablyforms a channel gate in the membrane. Such a dynamic process is expected torequire proper association of the subunits in multimeric Kir channels. Usingthe Kir6. 2 channel as a model, we have identified a region (234 through 243)on the Oterminus of kir6.2 that is likely involved in subunit-subunitinteraction which can change Kir6. 2 gating. We have analyzed the functionalsignificance of intersubunit association in the cytoplasmic domain throughmutagenic perturbation of a putative intersubunit interface area at His-234^ Gly-243. In the absence of the channel inhibitor ATP, mutations in thisarea at Val²36 caused the channel to lose activity, which was transientlyrestored fol lowing application then subsequent withdrawal of ATP. The effectof Val-236 mutations corresponded to the Van der Waals volumes of thesubstituents; with alanine, serine, and cysteine inhibit channel activity,and leucine and arginine having essentially no effect. Tn contrast to theVal-236 mutations, a double mutation at Asp-237 and Met-240 promoted channelactivity. Using an isolated recombinant cytoplasmic segment of Kir6.2, wehave further demonstrated that Val-236 mutations reduce the formation ofmultimers of the cytoplasmic segment, while the double mutation enhancesmultimer formation. The compromised oligomerization in Val-236 mutants wasimproved after ATP binding to the cytoplasmic segment (data not shown). Thus,channel activity measured in functional experiments parallels cytoplasmicintersubunit association assessed in biochemical experiments. A molecularmodel of the cytoplasmic domain by homo logy modeling based on the publishedcoordinates of other channels was constructed to interpret the mutagenes is.A kinetic model interpreting the effects of mutation is also given.Taken together, we infer that activation of Kir6.2 channels requires association of the cytoplasmic segments, and that compromise of this association results in the channel dwelling in an inactive state with channel subunit-subunit disassociated. Intersubunit interaction can weaken...