ABSTRACT
The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Calcium Channels, L-Type/metabolism , Calcium/metabolism , Receptors, Antigen, T-Cell/metabolism , Alternative Splicing , Animals , CD4-Positive T-Lymphocytes/cytology , Calcium Channels, L-Type/genetics , Exons , HEK293 Cells , Humans , Mice , RNA SplicingABSTRACT
We have used a microcontact printing approach to produce high quality and inexpensive holey carbon micro-arrays. Fabrication involves: (1) micromolding a poly(dimethylsiloxane) (PDMS) elastomer stamp from a microfabricated master that contains the desired array pattern; (2) using the PDMS stamp for microcontact printing a thin sacrificial plastic film that contains an array of holes; (3) floating the plastic film onto TEM grids; (4) evaporating carbon onto the plastic film and (5) removing the sacrificial plastic film. The final holey carbon micro-arrays are ready for use as support films in TEM applications with the fidelity of the original microfabricated pattern. This approach is cost effective as both the master and the stamps have long-term reusability. Arbitrary array patterns can be made with microfabricated masters made through a single-step photolithographic process.
Subject(s)
Microscopy, Electron/methods , Carbon , Dimethylpolysiloxanes , Microscopy, Atomic Force , Microscopy, Electron/instrumentation , Microscopy, Electron, Scanning , Nanotechnology/instrumentation , Nanotechnology/methods , Surface PropertiesABSTRACT
We present a microfluidic system integrated with disposable cell interface partitions for simultaneous patch clamp recordings. Glass-supported poly(dimethylsiloxane) (PDMS) partitions, having a 2 microm air-blown aperture, were reversibly sealed to a microfluidic system including PDMS channels with isolation valves and microfabricated Ag/AgCl electrodes. Gigaseal recordings from RBL-1 cells were obtained with a 24% success rate. Simultaneous whole cell recordings from valve-isolated electrodes were obtained.
Subject(s)
Cell Culture Techniques/instrumentation , Membrane Potentials/physiology , Microelectrodes , Microfluidic Analytical Techniques/instrumentation , Nanotechnology/instrumentation , Patch-Clamp Techniques/instrumentation , Animals , Cell Culture Techniques/methods , Cell Line , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/methods , Mast Cells/physiology , Microfluidic Analytical Techniques/methods , Nanotechnology/methods , Patch-Clamp Techniques/methods , RatsABSTRACT
The electrical activity of living cells can be monitored in various ways, but for the study of ion channels and the drugs that affect them, the patch-clamp techniques are the most sensitive. Recent developments in microfabricated patch-clamp electrodes are reviewed, and technical challenges for the future are discussed.
Subject(s)
Action Potentials/physiology , Cell Culture Techniques/instrumentation , Ion Channel Gating/physiology , Ion Channels/physiology , Lab-On-A-Chip Devices , Membrane Potentials/physiology , Patch-Clamp Techniques/instrumentation , Cell Culture Techniques/methods , Cell Culture Techniques/trends , Equipment Design , Ion Channels/chemistry , Microchip Analytical Procedures/methods , Nanotechnology/instrumentation , Nanotechnology/methods , Patch-Clamp Techniques/methods , Patch-Clamp Techniques/trends , Research/instrumentation , Research DesignABSTRACT
We present a new technique for fabricating planar patch electrodes in the laboratory. Planar electrodes are micromolded using a micron-sized stream of air to define an aperture in the silicone elastomer, polydimethylsiloxane (PDMS). We have previously demonstrated that planar PDMS electrodes make excellent patch electrodes after surface modification. We demonstrate single-channel measurements of the rSlo channel in Xenopus oocytes and whole-cell measurements in CHO and RBL mammalian cell lines, using planar PDMS electrodes.
Subject(s)
Microelectrodes , Patch-Clamp Techniques/instrumentation , Patch-Clamp Techniques/methods , Air , Animals , CHO Cells , Cricetinae , Equipment Design , Female , Membrane Potentials/physiology , Oocytes/physiology , Xenopus laevisABSTRACT
The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.
