Dispensable ATP permeability of Pannexin 1 channels in a heterologous system and in mammalian taste cells

Abstract

SummaryAfferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.

The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer

“…Our findings indeed support the lack of proper intracellular localization of connexin43 expression in PC-3 cells, but clearly indicate the existence of functional GJICs in these cells by dye-transfer experiments, which we believe are comprised at least in part of the pannexin family of proteins [28,29]. Bystander cell killing effect was completely abolished with the physical separation of the effector and bystander cells in transwell experiments as well as upon GJIC inhibitor treatment, highlighting the requirement for functional GJICs and indicating that the bystander effects observed in the TMPK/AZT system are not mediated by any soluble factor or free diffusion of antimetabolites to bystander cells.…”

A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction

“…For these experiments, we made use of 293T cells, devoid of endogenously expressed CBX-sensitive currents (45,46), and we assessed pannexin function by recording membrane currents generated by voltage ramps from Ϫ100 to ϩ100 mV. In 293T cells expressing R217H, ramp currents recorded at ϩ100 mV were reduced by ϳ50% compared with wild-type PANX1 (Fig.…”

“…The pannexin family of channel-forming glycoproteins has grown in importance given their documented roles in ischemia, stroke, overactive bladder, HIV infections, Crohn's disease, platelet aggregation, and over a half-dozen other diseases (13,28,45,48,49). In most of these cases, PANX1 was identified as being the key pannexin linked to the disease, but the causal mechanism of how PANX1 large-pore channels are associated (bottom).…”

Chemotherapeutic Drugs Induce ATP Release via Caspase-gated Pannexin-1 Channels and a Caspase/Pannexin-1-independent Mechanism

“…Six Panx1 subunits co-assemble into stable hexameric channels during synthesis in the endoplasmic reticulum/Golgi network and trafficking to the plasma membrane. In the basal state, Panx1 channels are defined by very low open probability and conductance (10,(12)(13)(14)(15)(16). Increased Panx1 channel activity in the plasma membrane of nonapoptotic cells can be rapidly (within seconds to minutes) induced by mechanical stresses, stimulation of some G protein-coupled receptors, or strong depolarization of the membrane potential to Ͼ0 mV (17).…”