Molecular Electroporation and the Transduction of Oligoarginines

📝 Abstract

Certain short polycations, such as TAT and polyarginine, rapidly pass through the plasma membranes of mammalian cells by an unknown mechanism called transduction as well as by endocytosis and macropinocytosis. These cell-penetrating peptides (CPPs) promise to be medically useful when fused to biologically active peptides. I offer a simple model in which one or more CPPs and the phosphatidylserines of the inner leaflet form a kind of capacitor with a voltage in excess of 180 mV, high enough to create a molecular electropore. The model is consistent with an empirical upper limit on the cargo peptide of 40–60 amino acids and with experimental data on how the transduction of a polyarginine-fluorophore into mouse C2C12 myoblasts depends on the number of arginines in the CPP and on the CPP concentration. The model makes three testable predictions.

💡 Analysis

Certain short polycations, such as TAT and polyarginine, rapidly pass through the plasma membranes of mammalian cells by an unknown mechanism called transduction as well as by endocytosis and macropinocytosis. These cell-penetrating peptides (CPPs) promise to be medically useful when fused to biologically active peptides. I offer a simple model in which one or more CPPs and the phosphatidylserines of the inner leaflet form a kind of capacitor with a voltage in excess of 180 mV, high enough to create a molecular electropore. The model is consistent with an empirical upper limit on the cargo peptide of 40–60 amino acids and with experimental data on how the transduction of a polyarginine-fluorophore into mouse C2C12 myoblasts depends on the number of arginines in the CPP and on the CPP concentration. The model makes three testable predictions.

📄 Content

Molecular Electroporation and the Transduction of Oligoarginines Kevin Cahill November 18, 2018 cahill@unm.edu Biophysics Group, Department of Physics & Astronomy, University of New Mexico, Albuquerque, NM 87131 ABSTRACT Certain short polycations, such as TAT and polyarginine, rapidly pass through the plasma membranes of mammalian cells by an unknown mechanism called transduction as well as by endocytosis and macropinocytosis. These cell-penetrating peptides (CPPs) promise to be medically useful when fused to biologically active peptides. I offer a simple model in which one or more CPPs and the phosphatidylser- ines of the inner leaflet form a kind of capacitor with a voltage in excess of 180 mV, high enough to create a molecular electropore. The model is consistent with an empirical upper limit on the cargo peptide of 40–60 amino acids and with experimental data on how the transduction of a polyarginine-fluorophore into mouse C2C12 myoblasts depends on the number of arginines in the CPP and on the CPP concentration. The model makes three testable predictions. I. CELL-PENETRATING PEPTIDES In 1988, two groups [1, 2] working on HIV reported that the trans-activating transcriptional activator (TAT) of HIV-1 can cross cell membranes. The engine driving this 86-aa cell-penetrating peptide (CPP) is its residues 48–57 grkkrrqrrr which carry a charge of +8e. Other CPPs soon were found. Antp (aka Penetratin, PEN) is residues 43–58 rqikiwfqnrrmkwkk of Antennape- dia, a homeodomain of the fly; it carries a charge of +7e. The polyarginine (Arg)n carries charge +ne, where often n = 7, 8, or 9. Other CPPs have been discov- ered (VP22) or synthesized (transportan). The struc- tural protein VP22 of the tegument of herpes simplex virus type 1 (HSV-1) has charge +15e. Transportan gwtlnsagyllg-k-inlkalaalakkil-amide is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan and a connecting lysine [3]. With its ter- minal amide group, its charge is +5e. These and other short, positively charged peptides can penetrate the plasma membranes of live cells and can tow along with them cargoes that greatly exceed the 600 Da restriction barrier. They are promising therapeutic tools when towing cleverly chosen peptide cargoes of from 8 to 33 amino acids [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. Many early experiments on CPPs were wrong be- cause the cells were fixed or insufficiently washed. Even careful experiments sometimes have yielded inconsistent results—in part because fluorescence varies with the (sub)cellular conditions and the fluorophores [17]. Yet some clarity is emerging: TAT carries cargoes across cell membranes with high efficiency by at least two functionally distinct mechanisms according to whether the cargo is big or small [18]. Big cargoes, such as pro- teins or quantum dots, enter via caveolae endocytosis and macropinocytosis [19, 20], and relatively few escape the cytoplasmic vesicles in which they then are trapped [18]. Small cargoes, such as peptides of fewer than 30– 40 amino acids, enter both slowly by endocytosis and rapidly by transduction with direct access to the cytosol, an unknown mechanism that uses the membrane poten- tial [18, 21, 22, 23, 24]. Peptides fused to TAT enter cells within seconds [25]. It remains unclear how big cargoes aided by several CPPs enter cells [26]. For instance, superparamegnetic nanoparticles encased in aminated dextran and attached to 45 tat peptides are thought to enter cells by adsorptive endocytosis[27, 28, 29] but they do enter slowly at 4◦ C [30]. This paper is exclusively about how polycationic cell- penetrating peptides, specifically oligoarginines, trans- duce small cargoes directly into the cytosol. Sec. II recalls some basic facts about plasma membranes, and Sec. III explains why ions do not normally pass through plasma membranes. Sec. IV describes a simple model of the transduction of CPPs in which electroporation and phos- phatidylserine play key roles. In this model, one or more positively charged CPPs on the outer leaflet and the neg- atively charged PSs under it on the inner leaflet form a kind of capacitor, which enhances the membrane poten- tial to a voltage in excess of 180 mV, which is sufficient to create an electropore. Sec. V shows that the model is consistent with an empirical upper limit on the cargo of 40–60 amino acids and with measurements made by T¨unnemann et al. [31] on the fraction of mouse myoblasts transduced by polyarginines carrying fluorophores of 400 Da. Sec. VI tells how to test three predictions of the model. The paper ends with a short summary in Sec. VII. II. MAMMALIAN PLASMA MEMBRANES The plasma membrane of a mammalian cell is a lipid bilayer that is 4 or 5 nm thick. Of the four main phos- pholipids in it, three—phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM)—are neutral, and one, phosphatidylserine (PS), is negatively charged. In live ce

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