Nonselective cationic currents elicited by extracellular ATP in human B-lymphocytes

Abstract

Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of Ip.

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…We calculated similar ratios (ϳ2-3) for the native responses of mouse and human macrophages and lymphocytes. All of these values are comparable with those measured from other types of ligand-gated ion channels (82), but remarkably different from that (P Ca /P Na ϭ 35) reported in an early study (92) of the unclassified (but presumably P2X7R-mediated) ATP-gated current of transformed human B-lymphocytes. Although it is possible that this ATP- FIGURE 8.…”

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…Although the role of alternative splicing of the P2X receptor is still unclear, studies have demonstrated that some mis-spliced variants are synthesized as truncated proteins that can interact with full-length receptors to regulate their function [71,90,91]. Unlike the other P2X receptors (P2X [1][2][3][4][5][6] ), P2X 7 is usually considered to form fully functional homotrimers, although evidence for functional P2X 4 /P2X 7 heteromeric receptors has also been reported [97]. Interaction of truncated P2X 7 proteins with full-length P2X 7 through hetero-oligomerization may represent a general paradigm for regulation of protein function by a variant protein encoded by the same gene.…”

“…All of the P2X receptors contain two membrane-spanning domains, intracellular amino and carboxy termini, and an extracellular domain of approximately 280 amino acids containing the ligand-binding site and ten conserved cysteine residues that likely form intrachain disulfide bonds to give secondary structure to the receptor [2][3][4][5]. P2X 7 receptors differ from other P2X receptor subtypes (P2X [1][2][3][4][5][6] ) by an extended cytoplasmic carboxy-terminal tail (240 aa) which may be responsible for the unique ability of P2X 7 to form large pores in the membrane after prolonged agonist stimulation [1]. The P2X 7 receptor exhibits several unusual properties.…”

“…The P2X 7 receptor exhibits several unusual properties. Following brief activation by agonist, P2X 7 forms a channel with strong selectivity for the divalent cations Ca 2+ and Ba 2+ over monovalent cations [6]. Continued stimulation by agonist results in the formation of a non-selective pore that allows permeation of inorganic and organic cationic molecules up to 900 Da, such as ethidium bromide, propidium iodide (PI), and quinolinium fluorescent dyes YO-PRO-1 [4,7].…”

Extracellular ATP in the lymphohematopoietic system: P2Z purinoceptors and membrane permeabilization

“…Plasma membranes of cells of the lymphohematopoietic system can be routinely patch-clamped, providing a high-resolution time-resolved description of many events that follow receptor-ligand interaction. Recently, a better understanding of the properties of the P2Z purinoceptor has been achieved by applying these techniques to macrophages and other cells (9,18,29,(32)(33)(34)(35)(36)(37)(38).…”