Facilitation of P2X7 Receptor Currents and Membrane Blebbing Via Constitutive and Dynamic Calmodulin Binding

The ATP-gated P2X(7) receptor (P2X(7)R) is a highly unusual calcium-permeable cationic channel in that within seconds of its activation, dramatic and reversible cytoskeletal rearrangements with prominent membrane blebbing occurs. Agonist-induced membrane currents at hyperpolarized potentials show pronounced facilitation during the initial 30-100 s of receptor activation but mechanisms responsible have not been elucidated. We measured facilitation of ATP-gated currents in HEK cells expressing rat P2X(7)R and delineated distinct calcium-dependent and independent processes. The calcium-dependent facilitation was composed of an instantaneous (millisecond time domain) and slowly developing (time constant, 20 s with maximum agonist stimulation) component. Both components were prevented when recording with a highly specific calmodulin (CaM) inhibitory peptide but only the instantaneous component was reduced by expression of the dominant-negative EF-handless CaM mutant. Coimmunoprecipitation assays detected low levels of CaM binding to unstimulated P2X(7)R, and this increased by 50% during 45 s stimulation of the receptor. We identified a novel 1-5-16 Ca(2+)-dependent CaM binding motif in the intracellular C terminus of P2X(7)R; mutations in this domain resulted in the absence of calcium-dependent facilitation and binding of CaM to unstimulated or stimulated receptor. Blockade of CaM binding also delayed membrane blebbing by threefold. Our results demonstrate that CaM binds constitutively to closed P2X(7)R channels and dynamically during channel activation to significantly enhance and prolong calcium entry. This is the first example of CaM deregulating, rather than tightly controlling, calcium entry through an ion channel.