ABSTRACT
Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 ß-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.
Subject(s)
Potassium Channels, Voltage-Gated/chemistry , Animals , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetinae , Cricetulus , Detergents/chemistry , Humans , Microscopy, Electron , Patch-Clamp Techniques , Polystyrenes/chemistry , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SolubilityABSTRACT
OBJECTIVE: To increase awareness of the possibility of epidural infection after continuous epidural infusion. Outline the salient diagnostic features of epidural infection. Outline a strategy to manage epidural infection and minimize morbidity. SETTING: Academic multidisciplinary pain clinic. PATIENT: A patient with a left knee meniscal tear with a history of Chronic Regional Pain Syndrome Type I (CRPS I) of the left foot. INTERVENTIONS: Attempted control of CRPS I symptoms with a tunnelled epidural catheter infusion. RESULTS AND CONCLUSIONS: The patient developed an epidural abscess diagnosed on the 11th postoperative day. The catheter was removed and the patient was treated successfully with intravenous antibiotics.
