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Methods Mol Biol
2024 Jan 01;2799:151-175. doi: 10.1007/978-1-0716-3830-9_9.
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Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.
Iacobucci GJ
,
Popescu GK
.
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In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.
Fig. 1Potential sources of experimental and statistical errors. (a) Example of classic “J-shape” <i>/V relationship indicates the presence of contaminating voltage-dependent blockers (usually divalent metal ions, M2+), which can be eliminated with a metal-chelating buffer. (b) Top, fraction of free buffer available to capture contaminating metals in each total added Ca2+. Below, predicted free concentrations of contaminating metals, given predicted free concentrations of each indicated buffer at various total added Ca2+. (c) Leak-subtracted, average unitary current in 2 mM Ca2+ (gray) overlaid with fits of the linear model (yellow) and the Jahr model (red) to account for nonlinearity of <i>/V relationship
Fig. 2Macroscopic and unitary conductance measurement. (a) Top, schematic of applied voltage (red), glutamate (1 mM Glu, black), and glycine (0.1 mM Gly, gray). Bottom, macroscopic current elicited with the protocol above from a HEK293 cell expressing GluN1/GluN2A recombinant receptors using whole-cell patch clamp. Upon Glu-application, the current rises from a zero level to a peak level (Ipk), reflecting receptor activation (yellow), after which the current declines gradually, indicative of desensitization (green), to a steady-state level (Iss) (green). At steady state, when the Po is constant, the applied voltage is ramped from −100 mV to +60 mV and then returned to −100 mV. Upon Glu withdrawal, the current declines rapidly to the initial zero level, indicative of deactivation (blue). (b) Unitary current trace recorded in the continuous presence of 1 mM Glu and 0.1 mM Gly using cell-attached path-clamp reflects the steady-state phase of the macroscopic current, when channels maintain constant Po by cycling stochastically through periods of activation, deactivation, and desensitization. (c) Current trace recorded form a cell-attached membrane patch illustrates three characteristic current levels indicative of zero, one, and two simultaneously open channels. (d) The probability that a recording with openings to only one level originates from a patch with more than one active channel decreases with the number of consecutive openings observed (no) and with the Po of the channels observed
Fig. 3Recording unitary current during a voltage ramp. (a) Top, voltage ramp protocol used to elicit unitary currents from receptors residing in a cell-attached patch. Bottom, current traces recorded from a cell-attached patch subjected to consecutive sweeps of the protocol above (gray). Sweeps 2 and n elicit no openings, indicative of a desensitized period (red). (b) Left, observed current (<iobs>) represents the average of all sweeps (black). Right, observed leak current <ileak > represents the average of all sweeps with no openings
Fig. 4Correcting voltage-ramp currents for channel kinetics. (a) In physiologic conditions, single-channel recordings illustrate reduced global open probability (Po), reduced principal current level (Op), and a prominent subconductance level (Os) relative to 0 Ca2+ conditions. (b) Unitary currents recorded in the presence of increasing extracellular Ca2+ concentrations. (c) Top, leak-subtracted, average unitary current (Fig. 3). Below, resultant leak-subtracted unitary current <i> corrected for channel open probability which varies with Ca2+. (d) Leak-subtracted, Po-corrected unitary currents recorded during voltage ramp at several extracellular Ca2+ concentrations
Fig. 5 Voltage-step method for measuring unitary conductance. (a) Unitary currents recorded using cell-attached patch clamp at various applied voltages in the absence (left) and presence (right) of extracellular Ca2+. Dashed lines indicate zero-current amplitudes for closed channels (C) and two levels of nonzero current for the principal (Op) and the subconductance (Os) level. (b) Unfiltered, raw unitary current traces recorded at several applied voltages with corresponding amplitude histograms. Curves represent conductance-level class prediction using segmental-k-means algorithm. (c) Current-voltage relationships recorded in zero and 2 mM Ca2+
