Functional studies of purified recombinant BK-CA channels and the mechanosensitive channel of high conductance (MscL)) reconstituted in bilayer lipid membranes tethered to a microelectrode array device

The measurement of single-channel activity of the high conductance calcium-activated potassium (BKCa) channels incorporated in tethered bilayer lipid membranes is a major step towards the development of biosensors based on ion channels. Recombinant BKCa channels were expressed with hexa-histidine fusion tags and isolated from Xenopus laevis oocyte membranes by detergent solubilization. The histidine tag was included to facilitate purification of the desired channels by immobilized metal-ion affinity chromatography, which provided a convenient purification protocol that yielded high purity BKCa channels which in turn were successfully reconstituted in vesicles consisting of phytanoyl phospholipids.;Biophysical characterization of vesicular and solid-supported bilayer lipid membrane assemblies of two different phytanoyl lipids, 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl- sn-glycero-3-phosphoethanolamine (DPhPE) were performed. These studies revealed the appropriate physical conditions and optimal lipid concentrations required to form stable membranes suitable for both reconstitution of proteins and for tethering on solid substrates to form electrically stable membranes. Results showed that the most stable biomimetic model membranes were formed using a combination of DPhPC and DPhPE lipids in a 7:3 ratio; these were then interfaced to microelectrode array devices for incorporation of channels for functional studies. Formation of the tethered bilayer lipid membrane on a gold substrate and the successful incorporation of BKCa channels in this membrane system were verified with surface plasmon resonance-enhanced ellipsometry.;Functional studies of incorporated channels were performed using a modified patch clamp electrophysiology technique and single-channel events were recorded. Results indicate that the IMAC purified BKCa channels were functional and that their electrical properties in the tBLM system had similarities with channels investigated under conventional patch-clamp conditions. However, conductance levels were lower in the tBLM and channels in this membrane system exhibited slower gating kinetics. Pharmacological studies of tBLM incorporated BKCa channels showed that these channels were sensitive to tetraethylammonium compounds at micromolar concentrations, just like wild type channels investigated by patch clamp techniques.;The mechanosensitive channel of large conductance (MscL), a stretch-activated ion channel isolated from E. coli was also studied in this experimental set-up. This study represents the first documented report on the investigation of MscL in a supported bilayer membrane. Results obtained here demonstrate that the MscL channel can be activated by voltage and that the channel is gated in response to stress in the lipid membrane as opposed to pressure across it. Furthermore, these findings show the possibility of using MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.