Activation of Calcium-activated Potassium Channel Following Stimulation of P2X7 Receptors in Retinal Ganglion Cells

Abstract

Purpose: : Mechanical strain induces an increase of extracellular ATP in the retina. This ATP can activate ionotropic P2X₇ receptors on retinal ganglion cells (RGCs), leading to the influx of Na⁺ and Ca²⁺. The increased intracellular Ca²⁺ may in turn open calcium-activated K⁺ (KCa) channels. As the opening of KCa channels would hyperpolarize the membrane and reduce the spike output to the optic nerve, this interaction could impact visual signaling. This study thus asks if P2X₇ receptor stimulation leads to KCa channel activation in RGCs.

Methods: : KCa currents in RGCs from rat mixed neonatal retina cultures were measured using standard patch clamp protocols. RGCs, identified in mixed culture after injection of fluorescent label into the superior colliculus, were voltage clamped from -100 mV to +80mV for 300 ms from a holding potential of -60mV. Pipette solution contained no added Ca²⁺ and 0.1 mM EGTA. BzATP (100 µM) was used to activate the P2X₇ receptor. Specific blockers apamin (200 nM) and iberiotoxin (30 nM) were added once a stable current was achieved to determine the contribution from small and large conductance KCa channels respectively.

Results: : P2X₇ receptor agonist BzATP activated a robust current in 75% of RGCs. Currents reached a peak 4.1 min after exposure to BzATP and displayed a mild outward rectification. Apamin reduced the BzATP-activated current in all cells tested with maximum block found within 6 min of first exposure. The inhibition by apamin was voltage independent and produced a 69±12% block (p<0.05, n=4). Iberiotoxin also inhibited the currents in four out of five cells tested within 6 min, producing a near complete block (p<0.05, n=4).

Conclusions: : These results suggest that activation of the P2X₇ receptor in RGCs may have secondary effects on KCa channels. Blockade by apamin implicates the small conductance KCa channels while blockade by iberiotoxin suggests an involvement of large conductance subtypes. The P2X₇ receptor is relatively permeable to Ca²⁺, in addition to activating L-type Ca²⁺ channels, so either pathway could lead to KCa channel activation. Whether mechanical stimulation can also activate KCa in RGCs, and whether this alters neural transmission, need to be studied further.

Keywords: ganglion cells • ion channels • adenosine