One-channel Cell-attached Patch-clamp Recording

Abstract

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patchclamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechanosensitive channel PIEZO1. This method can provide data regarding the channel's conductance properties and the temporal sequence of openclosed conformations that make up the channel's activation mechanism, thus helping to understand their functions in health and disease. Video LinkThe video component of this article can be found at

Actions of Bupivacaine, a Widely Used Local Anesthetic, on NMDA Receptor Responses

“…Rat GluN1 (UO8261) and GluN2A (M91561) or GluN2B (M91562) subunits were expressed from pcDNA3.1. Plasmids were transiently transfected at a GluN1:GluN2:GFP ratio of 1:1:1 using the calcium phosphate method (Chen and Okayama, 1987;Kussius et al, 2009). After 2 h of incubation, the transfection mixture was replaced with DMEM supplemented with 2 mM Mg 2ϩ to prevent excitotoxicity.…”

“…Cell-attached one-channel currents were recorded with fire polished pipettes (12-25 M⍀) containing the following (in mM): 1 glutamate, 0.1 glycine, 150 NaCl, 2.5 KCl, 10 HEPBS, 1 EDTA, without (control) or with bupivacaine, at pH 8.0 (with NaOH) (Maki et al, 2014). Inward sodium currents were amplified and low-pass filtered at 10 kHz with an applied pipette potential of 100 mV with Axopatch 200B (Molecular Devices), sampled at 40 kHz (NIDAQ board), and written into digital files using QuB software (www.qub.buffalo.edu).…”

“…In addition to a prominent blocking effect on voltage-gated Na ϩ channels, local anesthetics also modulate many other neuronal channels (Hara et al, 1995;Scholz, 2002). Recent studies demonstrated that bupivacaine, the local anesthetic most commonly used for spinal, epidural, and caudal anesthesia, inhibits NMDA receptor currents, thus raising the possibility that this inhibition may account for some of its specific clinical effects (Nishizawa et al, 2002;Sugimoto et al, 2003;Hahnenkamp et al, 2006;Furutani et al, 2010).…”

“…Given that inhibiting NMDA receptor currents is an effective strategy in prevention and management of chronic pain syndromes (South et al, 2003;Rondon et al, 2010;Woolf, 2010), bupivacaine, which is safe and long-lasting, may represent a useful treatment of central sensitization resulting pathologies. NMDA receptors are heterotetramers of two GluN1 and two GluN2 subunits (Karakas and Furukawa, 2014;Lee et al, 2014). Each subunit contains ligand-and allosteric modulator-binding extracellular domains, a transmembrane domain composed of three membranespanning helices (M1, M3, M4) and a P-loop (M2), and a large intracellular domain.…”

“…Relative to Na ϩ channels, the topology of NMDA receptors is reversed, with an external ligand-controlled gate, and the narrowest region of the pore located two-thirds into the membrane field. The permeation pathway is lined by residues on M3 helices and M2 loops of each subunit, and these form binding sites for divalent cations (Nowak et al, 1984) and other blocking molecules (Huettner and Bean, 1988;Bormann, 1989;Parsons et al, 1993;Sobolevsky et al, 1999). NMDA receptor currents can be reduced by pore blockers, which obstruct the permeation pathway in a voltage-dependent manner, and by allosteric modulators, which alter channel gating kinetics (Kawajiri and Dingledine, 1993;Popescu, 2005;Traynelis et al, 2010).…”