Reconstituted channels

Ion channels have been purified from several types of excitable cells. The proteins that make up the voltage-gated Na+ channels of brain neurons were first identified by labeling them with reactive derivatives of neurotoxins obtained from scorpions. Intact channels were purified from both brain and muscle after solubilization with detergents. When the purified proteins were incorporated into phospholipid vesicles or planar bilayer membranes, they were found to conduct Na+ across the membrane. The ion specificity of the reconstituted channels and the alterations of the conductance in response to changes in Aift or to various neurotoxins were similar to the properties of the original nerve or muscle membranes. In addition to scorpion toxins, a variety of other specific neurotoxins bind to the purified channels and inhibit their activities. These include tetrodotoxin (a poison obtained from... 

The studies outlined in this section describe the ways we have addressed the foregoing problems of connexin reconstitution by utilizing connexin-32, the predominant form of connexin in rat liver. Our goals were to establish unambiguously that connexin-32 formed channels in liposome membranes, to identify connexin channels in planar bilayers, and to study their properties. Two methods were used to identify reconstituted channels formed by connexin-32. In one method, protein was solubilized from preparations of junctional membrane and incorporated into unilamellar liposomes. Connexin-32 was identified as a channel-forming protein by its specific enrichment in liposomes that were permeable to sucrose. In the other method, connexin-32 was affinity-purified (with a monoclonal antibody directed specifically against connexin-32) directly from octylglucoside-solubilized plasma membranes. Liposomes formed with such material were permeable to sucrose and Lucifer Yellow. Sucrose-permeable liposomes from each method were fused with planar bilayers to study the properties of connexin channels. 

OmpF was successfully reconstituted into Upid vesicles (186,218), which could be demonstrated using an encapsulated enzyme, -lactamase, which is able to hydrolyze the antibiotic ampicillin. The hydrolysis product, ampicillinoic acid, can reduce iodine to iodide, which can be monitored by iodometry, ie via the decol-orization of a starch/iodine complex in the exterior solution. The enzyme and the membrane channel have been shown to preserve their activity in the presence of hydrophobic methacrylate monomers before and after their cross-linking polymerization. Polymerization of hydrophobic iV-butylmethacrylate and ethylene glycol dimethacrylate (186) lead to partial expulsion of some reconstituted channels during the cross-linking reaction, thereby decreasing the enzymatic activity. 

Lai R, Kim FI, Garavito R M and Arnsdorf M F 1993 Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy Am. J. Physiol. 265 C851... 

Extraction of Sodium Channel Blockers. A review of published reports shows that methods for purification of sodium channel blockers from bacterial cultures are similar to techniques for isolation of TTX and STX from pufferfish and dinoflagellates (30, 31, 38, 39). Typically, cell pellets of bacterial cultures are extracted with hot 0.1% acetic acid, the resulting supernatant ultra-filtered, lyo-philized, and reconstituted in a minimal volume of 0.1% acetic acid. Culture media can also be extracted for TTX by a similar procedure (Ji). Both cell and supernatant extracts are analyzed further by gel filtration chromatography and other biological, chemical, and immunological methods. Few reports describe purification schemes that include extraction of control samples of bacteriological media (e.g., broths and agars) which may be derived from marine plant and animal tissues. 

The ion channel receptors are relatively simple in functional terms because the primary response to receptor activation is generated by the ion channel which is an integral part of the protein. Therefore, no accessory proteins are needed to observe the response to nicotinic AChR activation and the full functioning of the receptor can be observed by isolating and purifying the protein biochemically and reconstituting the protein in an artificial lipid membrane. In contrast, the G-protein-coupled receptors require both G-proteins and those elements such as phospholipase-C illustrated in Fig. 3.1, in order to observe the response to receptor activation (in this case a rise in intracellular calcium concentration resulting from the action of IP3 on intracellular calcium stores). 

Reconstitution of Ca channels Since L-type Ca channels were purified as high affinity DHP receptors, it was important to demonstrate that the purified preparations could act as functional DHP-sensitive Ca channels. Purified DHP receptor preparations from skeletal... 

The recent studies with the skeletal muscle channels reconstituted in bilayers also established that an intrinsic property of the skeletal muscle Ca channels is that they possess a very low probability of opening. Even under optimal conditions of voltage, etc., these channels open only 8% of the time [95]. This previously unappreciated characteristic of these channels provides an alternative explanation to an earlier study in which it was proposed that only 5% of the DHP receptors in skeletal muscle can form functional channels [91]. In that study a probability of opening of 1.0 was assumed. However, if one adjusts this to 0.08, then the very different conclusion is that all the DHP receptors can make functional channels, but that they open only a very small percentage of the time. 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

Using the reconstitution approaches described above, we have demonstrated that phosphorylation of the skeletal muscle Ca channels by PKC results in activation of the channels [108], In the fluo 3-containing liposomes, channels phosphorylated by PKC exhibited a two-fold increase in the rate and extent of Ca " influx [108], Using the lipid bilayer-T-tubule membrane reconstitution system we are currently analyzing the effects of PKC-catalyzed phosphorylation at the single channel level [133], The demonstration that these channels undergo phosphorylation as a result of activation of PKC in intact skeletal muscle cells has not yet been achieved. 

The skeletal muscle Ca channels also can be phosphorylated in vitro by a protein kinase endogenous to the T-tubule membranes [111,115]. This kinase is neither Ca - nor cyclic nucleotide-dependent [115], and is interesting in that it phosphorylates primarily the P subunit while the ai subunit is a poor substrate. However, the amount of this kinase that co-purifies with the T-tubule membranes is variable, and consequently, very few studies have been performed. So far, only low levels of phosphorylation have been obtained (no more than 0.2 mol phosphate/ mol P subunit) and no functional effects of this phosphorylation have been observed in reconstitution studies. 

The mechanisms of the oscillations in biomembranes have been explained based on the gating of membrane protein called an ion channel, and enormous efforts have been made to elucidate the gating process, mainly by reconstitution of channel proteins into bilayer membranes [9-11]. 

C. Miller, Ion Channel Reconstitution, Plenum Press, New York, 1986. 

A G-protein-mediated effect has an absolute requirement for GTP. Reference has already been made to the requirement for GTP in reconstituting hormone-stimulated adenylate cyclase activity. A similar requirement can be demonstrated when the effector is an ion channel, such as the cardiac atrial inward-rectifier K+ channel which is activated following stimulation of the M2 muscarinic acetylcholine receptor. Thus, in the experiment illustrated in Figure 7.8, the channel recorded with a cell-... 

---