Single channel properties of pannexin‐1 and connexin‐43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords

Abstract

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp.The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs.Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides

“…The representative traces depicted in Figure 1A (enlarged display in Supplementary Figure S1) show unitary Panx1 current activities evoked by repeated (every 40 s) V m steps from a holding potential of −30 mV to +70 mV (with a duration of 30 s) in voltage-clamp experiments (using whole-cell patch-clamp recording). Under control conditions, a single channel conductance of ~65 pS was observed as determined from the channel transition histograms shown in Figure 1B, i.e., below the 100 pS range, which is typical for voltagedependent Panx1 channel-opening activity [4][5][6]. We tested the effect of 10 Panx1 and Gap19 on these currents.…”

“…Current methods to distinguish between the two channels rely on electrophysiological approaches (mainly single-channel conductance measurements) and peptide-based inhibitors targeting Cx43 or Panx1. However, single-channel conductance analyses are not without ambiguity, since Cx43 HCs show a fully open conductance in the order of 200-240 pS and a substate of ~80 pS, while Panx1 channel conductance has been reported to be as high as 550 pS in exogenously expressing Xenopus oocytes and between 25 and 100 pS in mammalian cells [4][5][6]. A second and commonly used strategy is to employ peptides designed to mimic specific regions in the native sequence of connexins and pannexins to interfere with channel function/gating.…”

Age-Dependent Activation of Pannexin1 Function Contributes to the Development of Epileptogenesis in Autosomal Dominant Sleep-related Hypermotor Epilepsy Model Rats

“…It has been established that probenecid and carbenoxolone are non-peptide hemi channel inhibitors [2,[45][46][47]. Probenecid is a relatively selective pannexin1-hemichanne inhibitor [46,48], whereas carbenoxolone is a non-selective inhibitor for the pannexin1 hemichannel, connexin43-hemichannel, gap junctions, and other channels [12,[49][50][51][52][53][54].…”

“…The 2 ′ (3 ′ )-O-(4-benzoylbenzoyl)adenosine-5 ′ -triphosphate tri(triethylammonium) salt (BzATP: potent rat P2X7R agonist) [84,85], 2-(phenylthio)-N-[[tetrahydro-4-(4-phenyl-1piperazinyl)-2H-pyran-4-yl]methyl-3-pyridinecarboxamide (JNJ47965567: selective P2X7R antagonist) [86], TAT-Gap19 (Gap19: selective connexin43 inhibitor) [87], 10PANX (selective pannexin1 inhibitor) [31], probenecid (pannexin1 inhibitor) [31,47], and carbenoxolone (CBX: non-selective astroglial hemichannel inhibitor) [2] were obtained from Funakoshi (Tokyo, Japan).…”

Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats

“…Astroglial hemichannels containing connexin 43 and pannexin 1 are also activated by membrane depolarization [63]. Furthermore, the functions of P2X7R, hemichannels containing connexin 43 and pannexin 1, were detected by voltage clamp [64]. Based on these findings, to explore the effects of membrane depolarizations on astroglial hemichannel activity, the effects of artificial HFOs on astroglial ATP release were determined.…”