Two mechanisms for inward rectification of current flow through the purinoceptor P2X₂ class of ATP‐gated channels

Abstract

The ATP receptor subunit P2X2 was expressed in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. ATP‐activated currents were studied with two‐electrode voltage clamp recordings from oocytes, whole‐cell recordings from HEK 293 cells, and outside‐out patch clamp recordings from both cell types. The steady‐state current‐voltage (I‐V) relation showed profound inward rectification in all recording configurations. Recordings from outside‐out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch. Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast voltage jumps, instantaneous current jumps were followed by substantial relaxations to the steady state. The time course of the current relaxations could be fitted by single exponential functions. The instantaneous I‐V relation was less inwardly rectifying than the steady‐state I‐V relation; however, it was not linear. Single channel recordings indicated that the single channel conductance became smaller when the membrane potential became more positive. This decrease could quantitatively account for inward rectification of the instantaneous I‐V relation. We conclude that inward rectification of P2X2 is due to two mechanisms: voltage‐dependent gating and voltage dependence of the single channel conductance.

ATP-mediated potassium recycling in the cochlear supporting cells

“…S1). This is also consistent with previous reports that inward currents pass more readily than outward currents through the P2X receptors, a characteristic referred to as inward rectification [32,33].…”

Molecular Physiology of P2X Receptors

“…This decline was not seen in calcium-free external solution. In outside-out patches, currents at P2X 2 receptors decline much more rapidly than in whole cell configuration; with normal extracellular calcium (1 mM) this decline occurs within tens of milliseconds (99,521). This basic observation implies that the decline of the current is prevented in the whole cell configuration because of the presence of some intracellular modulator, which is lost slowly in the whole cell recording but lost rapidly in outside-out patches (99).…”

Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

“…Under these conditions, the series resistance Extensive electrophysiological analyses of the P2X receptor channel have also been performed using heterologous expression systems, and several interesting features have been described. For instance, (1) all P2X channels are nonselective cation channels, and some are highly permeable to large cations ( Surprenant et al, 1996 ;Khakh et al, 1999 ;Virginio et al, 1999 ); (2) the macroscopic current shows inward rectifi cation, which is also observed in the unitary current ( Zhou and Hume, 1998 ;Fujiwara and Kubo, 2004 ); (3) upon opening after the application of ATP, some P2X channels show rapid desensitization, as other ligand-gated channels do, but P2X 2 , P2X 5 , and P2X 6 show very slow desensitization ( Ralevic and Burnstock, 1998 ;Smith et al, 1999 ;North, 2002 ;Fujiwara and Kubo, 2006b ); and (4) the functional properties of P2X channels are regulated by kinase activities ( Boue-Grabot et al, 2000 ;Hung et al, 2005 ), membrane lipids ( Elliott et al, 2005 ;Fujiwara and Kubo, 2006b ), and their expression density on the membrane ( Fujiwara and Kubo, 2004 ). Because the process of P2X 2 desensitization is slow, and channel activity persists for a considerable period after ATP application, the voltage dependence of the P2X 2 current can be analyzed using voltage step pulses in a manner similar to that used for voltage-gated channels.…”

“…3 D ) also showed similar tendencies. Because the single-channel current through the P2X 2 channel shows strong inward rectifi cation ( Zhou and Hume, 1998 ), it is necessary to analyze the G-V relationships of the deactivating tail current to estimate the channel activity (open probability of the channel) at each potential. The G-V relationships were analyzed by measuring the initial amplitudes of the tail currents at Ϫ 60 mV, and then fi tted with a two-state Boltzmann function.…”