Abstract
ATP-gated ionotropic P2X4 receptors are up-regulated in activated microglia and are critical for the development of neuropathic pain, a microglia-associated disorder. However, the nature of how plasma membrane P2X4 receptors are regulated in microglia is not fully understood. We used single-molecule imaging to track quantum dot-labeled P2X4 receptors to explore P2X4 receptor mobility in the processes of resting and activated microglia. We find that plasma membrane P2X4 receptor lateral mobility in resting microglial processes is largely random, consisting of mobile and slowly mobile receptors. Moreover, lateral mobility is P2X subunit- and cell-specific, increased in an ATP activation and calcium-dependent manner, and enhanced in activated microglia by the p38 MAPK pathway that selectively regulates slowly mobile receptors. Thus, our data indicate that P2X4 receptors are dynamically regulated mobile ATP sensors, sampling more of the plasma membrane in response to ATP and during the activated state of microglia that is associated with nervous system dysfunction.
Highlights
P2X4 receptors are ATP-gated ion channels that are expressed in microglia
C8-B4 microglia up-regulate expression of P2X4 receptors in the plasma membrane when they are challenged with LPS or fibronectin, which is reminiscent of P2X4 up-regulation within microglia in vivo (25)
The main findings of this study are as follows: (i) P2X4 receptors display distinct types of lateral mobility in microglia; (ii) their mobility increases in a use- and calcium-dependent manner in resting microglia, and (iii) their basal lateral mobility is under the control of p38 MAPK and significantly increased via this pathway in activated microglia
Summary
Results: P2X4 receptors in microglial processes display distinct types of lateral mobility that is regulated by ATP and by the activated state of microglia. ATP-gated ionotropic P2X4 receptors are up-regulated in activated microglia and are critical for the development of neuropathic pain, a microglia-associated disorder. Our data indicate that P2X4 receptors are dynamically regulated mobile ATP sensors, sampling more of the plasma membrane in response to ATP and during the activated state of microglia that is associated with nervous system dysfunction. Quantum dot (QD)3-based single-molecule imaging and single particle tracking (SPT) have shed light on the regulation of several neurotransmitter receptors, including P2X2 receptors (26, 31, 32), which are known to display different types of lateral mobility in the plasma membrane with proposed and demonstrated signaling roles (26). The lateral mobility of P2X4 receptors has not been measured, and little is known about this aspect of their function or even if mobility is regulated by ATP or in settings where P2X receptors are known to play critical roles, such as in resting and activated microglia
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