Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca2+
Ion channels are integral membrane proteins that regulate membrane potentials and signaling of cells in response to various stimuli. The patch-clamp technique enables the study of single channels or a population of channels. The macroscopic recording appr
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ntroduction Ion channels are integral membrane proteins that open or close transmembrane ion conduction pathways in response to various stimuli such as changes of temperature, pH, membrane potential, or ligand binding. By increasing or reducing membrane permeabilities to select ions, ion channels regulate membrane potentials and affect
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_10, © Springer Science+Business Media, LLC 2013
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signaling of cells. The important role of ion channels in many physiological processes has made ion channel recording techniques an important staple of many laboratories. Among the techniques that record ion channel activity, various configurations of the so called “patch-clamp technique” allow the recording of either populations of channel openings (macroscopic currents) or single channel openings. Both approaches offer advantages. Recordings of macroscopic currents offer insight into the cellular and average behavior of ion channels that reveal the magnitude of ionic currents and population response properties. Singlechannel recordings directly reveal individual ion channel behavior that underlies the macroscopic ion currents. Properties such as single-channel conductance, number of conformational states and rate constants, as well as heterogeneity of channels in the patch (both in terms of types and subunit compositions) can be directly evaluated using single-channel recordings (1). A disadvantage of single-channel recording is that analysis is more time consuming. One application of the single-channel recording technique is to provide accurate estimations of low steady-state open probabilities (Po < 0.01). Obtaining low Po data under conditions where certain aspects of gating can be investigated independently is very useful for understanding changes in gating mechanisms. This is in contrast to data from macroscopic recordings such as conductance– voltage (G–V) relationships of voltage-gated channels, where they describe channels’ steady-state behavior well, but is often not sufficient to achieve mechanistic understanding of channel gating. Single-channel recordings have been widely used to extend Po measurements in biophysical analysis of the BK type, largeconductance, Ca2+ and voltage-activated potassium channel (2–24). The BK channel gate can open at low probabilities in the absence of voltage-sensor activation and Ca2+ binding (intrinsic gating) (3, 25). Voltage-sensor activation and Ca2+ binding increase channel opening by allosteric coupling between the sensors and the gate (3, 25). Effects of modulators on these three aspects of BK channel gating: intrinsic-, voltage-, or Ca2+-dependent gating, are indistinguishable using G–V analysis alone. Simulations using a BK channel gating model (3) demonstrate this point (Fig. 1a). A tenfold increase in intrinsic gating and a −34 mV shift in voltage-sensor activation cause almost identical changes in the G–V relation. However
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