Teaching the modes of Ca2+ transport between the plasma membrane and endoplasmic reticulum using a classic paper by Kwan et al.

This teaching article uses the report by Kwan et al., “Effects of methacholine, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells,” where the effects of Ca2+-mobilizing agents in regulating Ca2+ fluxes were examined under various conditions. Upper-level undergraduate and new graduate students in physiology are the targe audience. Teaching and learning points are put forth in this article to illustrate 1) the characteristics of methacholine- and thapsigargin-induced Ca2+ responses, 2) the different endoplasmic reticulum Ca2+ stores accessible to methacholine and thapsigargin, 3) the inhibitory effects of La3+ on Ca2+ extrusion and Ca2+ influx, and 4) the facilitatory role of La3+ on endoplasmic reticulum Ca2+ recycling. Each of the above concepts is first explained with references to the figures adapted from the original article. A list of student learning questions then follows, where the answers are found in the teaching notes for the instructors. It is the objective of this article to make both teaching and learning Ca2+ regulation a rewarding experience for all.

Discovering the mechanism of capacitative calcium entry

This essay examines the historical significance of an APS classic paper that is freely available online: Kwan CY, Takemura H, Obie JF, Thastrup O, and Putney JW Jr. Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. Am J Physiol Cell Physiol 258: C1006–C1015, 1990. ( http://ajpcell.physiology.org/cgi/reprint/258/6/C1006 )

Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in LacrimalAcini

ABSTRACT The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to αv integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.