Paracrine signaling through plasma membrane hemichannels

Abstract

Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cell's interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1-2kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD(+) or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Fig. 1

P2X₇ receptors are essential to confer sensitivity to extracellular Ca²⁺ of Panx1 hemichannels. A. At normal extracellular Ca²⁺ concentration ([Ca²⁺]_o), P2X₇ receptors and Panx1 hemichannels both favor the closed states and no interaction takes place between these two channels. B. Removal of extracellular Ca²⁺ activates P2X₇ receptor by increasing the binding affinity (K_d lowering) of the channel for endogenously released ATP. Activation of the P2X₇ receptor initiates complex formation with Panx1 hemichannels which in turn serve as an associated portal, allowing the passage of ATP or reporter dyes with a MW below ~1 kDa. Lowering [Ca²⁺]_o may strengthen the interaction between P2X₇ receptor and Panx1 hemichannel further by an undefined signaling mechanism, yet independent on purinergic stimulation. Both P2X₇ receptor inhibitors such as Brilliant blue G and oxidized ATP and Panx1 hemichannel blockers carbenoxolone abolish the low [Ca²⁺]_o-triggered ATP release.

Fig. 2

Unitary current activities of Cx43 hemichannels. A. A schematic overview of whole cell recording in HeLa cells expressing Cx43 tagged with enhanced green fluorescent protein (EGFP) at the C-terminal. Currents mediated by Cx43 hemichannels are recorded under conditions of blockade of K⁺ channels using tetra-ammonium chloride (TEACl), BaCl₂ and CsCl. B. The fluorescent image of Cx43-EGFP cells, illustrates abundant presence of Cx43 at the plasma membrane. C. Representative whole-cell currents recorded in the Hela Cx43-EGFP cell indicated by an asterisk in panel B. Stepping V_m to +50 mV induces clearly discernible unitary current activities. The expanded trace shows tail currents with step-wise closure of 3 channels upon V_m repolarization to −25 mV. D. All-point histogram of the conductance evaluated from the trace shown in C at +50 mV. The peaks of the event distribution are separated by ~220 pS which equals to the γ_o of Cx43 hemichannels in the plasma membrane. E. Gently pulling the whole-cell recording electrode away from the cell establishes an outside-out recording configuration (extracellular face directed to the bath solution). F. Outside-out recordings often exhibit only a few active hemichannels as illustrated in the example current trace. G. All-point histogram of the conductance evaluated from the currents shown in E indicates a γ_o of ~220 pS.

Fig. 3

Role of Cx43 and Panx1 hemichannels in paracrine signaling in taste transduction. In taste buds, G-protein coupled receptors (GPCRs) for bitter, sweet and umami tastents are located at the apical membrane of type II receptor cells. Activation of the GPCR signaling pathway leads to the production of IP₃ and diacyl glycerol (DAG) through phospholipase Cβ (PLCβ)-mediated cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). An increase of [Ca²⁺]_i by IP₃-induced Ca²⁺ release opens transient receptor potential cation channel subfamily M member 5 (TRPM5). Na⁺ influx through TRPM5 channels depolarizes the cell membrane and stimulates voltage-gated Na⁺ channels, promoting the generation of an action potential (AP). Available evidence indicates that in response to tastent-evoked membrane depolarization, Cx43/Panx1 hemichannels open, facilitating ATP release. Extracellular ATP is thought to function as a neurotransmitter, targeting P2X₂/P2X₃R at postsynaptic afferent nerves. Alternatively, ATP may diffuse and bind to P2YR at adjacent presynaptic type III receptor cells. Following elevation of [Ca²⁺]_i by IP₃, a second taste transmitter 5-HT is secreted from the presynaptic cells through vesicular release.