Channel formation and [Ca2+]i accumulation induced by perforin N-terminus peptides: comparison with purified perforin and whole lytic granules.
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells express the pore forming protein perforin, which contributes to lymphocytotoxicity. The hallmark of perforin action is opening high-conductance transmembrane channels that enable massive influx of Ca2+ ions (deleterious to many cell types), as well as granzymes, which may trigger the apoptotic pathway. To explore the functional domains in the perforin molecule, we investigated in PN71 lymphocytes, the ability of perforin N-terminus synthetic peptides (compared to purified perforin and perforin-containing lytic granules), to cause intracellular Ca2+ ([Ca2+]i) accumulation and open transmembrane channels. To this end, we used the whole cell recording technique and Indo 1 fluorescence to measure membrane currents and [Ca2+]i, respectively. We have demonstrated that the N-terminus peptide Hu-34 (amino acids 1-34) closely resembled perforin action, reflected by [Ca2+]i accumulation and channel activity, while shorter peptides (e.g., Hu-16) generated mostly short-lived channels but no [Ca2+]i elevation. Hence, the first 34 amino acids of the perforin N-terminus sequence are sufficient for the perforin action.
Binah
O
O
Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty of Medicine, Haifa, Israel. binah@tx.technion.ac.il
Liu
C C
CC
Young
J D
JD
Berke
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000378:metabolism; D006825:Hybridomas; D007211:Indoles / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*
D015220:Calcium Channels; D005456:Fluorescent Dyes; D007211:Indoles; D007473:Ion Channels; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; C048960:indo-1; D002118:Calcium