Agonist-induced Ca ²⁺ entry determined by inositol 1,4,5-trisphosphate recognition

Abstract

It has been considered that Ca 2؉ release is the causal trigger for Ca 2؉ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP3R), the lack of Ca 2؉ entry in response to receptor activation is attributed to the absence of Ca 2؉ release. We reveal in this article that IP 3 R recognition of IP 3 determines agonist-induced Ca 2؉ entry (ACE), independent of its Ca 2؉ release activity. In DT40 IP3R ؊/؊ cells, endogenous ACE can be rescued with type 1 IP3R mutants (both a ⌬C-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca 2؉ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP3R-dependent manner without Ca 2؉ store release. Conversely, ACE cannot be rescued with mutant IP3Rs lacking IP3 binding (both the ⌬90 -110 and R265Q IP3-binding site mutants). We conclude that an IP3-dependent conformational change in the IP3R, not endoplasmic reticulum Ca 2؉ pool release, triggers ACE.C a 2ϩ transients elicited in response to cell surface receptor activation by neurotransmitters, hormones, and other molecular messengers are major messengers of intracellular communication (1). Stimulation of G protein-coupled receptors, tyrosine kinase receptors, and nonreceptor tyrosine kinases activate phospholipase C (PLC), catalyzing the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into the secondmessenger molecules: inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 mediates rapid Ca 2ϩ store release by activating IP 3 receptors (IP 3 Rs) in the endoplasmic reticulum (ER), whereas DAG activates protein kinase C (PKC) (2). After this initial Ca 2ϩ release phase, external Ca 2ϩ enters through plasma membrane channels, providing a secondary and more prolonged Ca 2ϩ signal (1), a phenomenon designated here as agonist-induced Ca 2ϩ entry (ACE) (3).To date, the molecular identity of these Ca 2ϩ entry channels as well as their coupling mechanism remain unknown, although several mechanisms have been proposed. Intracellular Ca 2ϩ release through the IP 3 R could trigger ACE by means of capacitative Ca 2ϩ entry (CCE) (4), which can be activated in a PLC-independent manner (3) by the ER Ca 2ϩ pump blocker thapsigargin or the Ca 2ϩ ionophore ionomycin (4, 5). In this scheme, the luminal drop in ER Ca 2ϩ activates ''store-operated'' Ca 2ϩ channels in the plasma membrane, although the basis for this coupling mechanism is entirely unknown. Although the release activity of IP 3 Rs may mediate Ca 2ϩ entry, others have suggested that IP 3 Rs play a conformational role in the coupling process (6, 7). Also, DAG may directly initiate ACE, because DAG can activate overexpressed ''canonical'' transient receptor potential Ca 2ϩ entry channels (TRPC) (8).We recently demonstrated a functional distinction between ACE and CCE based on the requirement of the former for PLC-␥, in a lipase-independent manner (3). However, it is unclear whether endogenous ACE is an integrated process accounting for both receptor an...

RACK1 binds to inositol 1,4,5-trisphosphate receptors and mediates Ca2+release

“…4B, red trace) almost doubles Ca 2ϩ release in response to purinergic receptor activation by UTP, although exerting no effect on subsequent agonist-induced Ca 2ϩ entry. These findings fit with previous evidence that IP 3 R-mediated Ca 2ϩ release and Ca 2ϩ entry are distinct processes (13). Depletion of RACK1 by siRNA profoundly reduces IP 3 R Ca 2ϩ release in response to UTP with no effect on subsequent Ca 2ϩ entry (Fig.…”

“…In all of these experiments, we observe no effects of RACK1 overexpression or its deletion on Ca 2ϩ entry (data not shown). This independent regulation of Ca 2ϩ entry and release accords with other studies (8,13).…”

Native TRPC7 Channel Activation by an Inositol Trisphosphate Receptor-dependent Mechanism

“…We found that a mutant DT40 cell line lacking all three IP 3 receptor isoforms (IP 3 R Ϫ/Ϫ cells) also lacked this PLC-dependent nonstore-operated cation entry, a finding more recently confirmed by others (9,10). If DT40 cells express only a single non-storeoperated entry involving TRPC7, one would expect that the lack of non-store-operated cation entry in IP 3 R Ϫ/Ϫ cells would be accompanied by absence of the OAG-regulated cation entry pathway.…”

“…Rather, in the avian B-cell line, TRPC7 appears to function as a PLC-regulated, DAG-activated non-selective cation channel. However, previous studies from this laboratory (8) and others (9,10) showed that in DT40 cells non-store-operated Ca 2ϩ , receptor-regulated cation entry depends in some manner on inositol trisphosphate (IP 3 ) receptors, but the precise role of IP 3 receptors (IP 3 R) in this pathway has not been defined. Because a Ca 2ϩ -impermeable mutant of the IP 3 R failed to rescue Ca 2ϩ entry in IP 3 R knock-out cells, we speculated that this entry might involve plasma membrane IP 3 R (8).…”

Modulation of store-operated Ca2+ entry by cyclic-ADP-ribose

“…It has been shown that agoniststimulated intracellular Ca 2+ transients are biphasic, with the initial, transient rise in Ca 2+ representing sarcoplasmic Ca 2+ release (1)(2)(3)(4), and the later sustained Ca 2+ response representing predominantly Ca 2+ influx (1)(2)(3)(4). One of the mechanisms of extracellular Ca 2+ influx is the so-called store-operated Ca 2+ entry (2)(3)(4)(5). The store-operated Ca 2+ entry (also called capacitative Ca 2+ entry) appears to be mediated by a large family of channels called the transient receptor potential ion channels (TRPC; [2][3][4][5].…”