Most inositol 1,4,5-trisphosphate receptors (IP 3 R) are expressed in the endoplasmic reticulum (ER), where their precise distribution underlies the spatially complex Ca 2؉ signals evoked by extracellular stimuli. The signals that target IP 3 R to the ER or, less commonly, to other membranes are unknown. We expressed yellow fluorescent protein-tagged fragments of type 1 IP 3 R alone or fused with a plasma membrane protein to establish the determinants of ER targeting in COS-7 cells. By using a combination of confocal imaging and glycoprotein analyses, we demonstrated that any pair of the six transmembrane domains (TMD) linked by a luminal loop retains the protein within the ER, and when attached to a plasma membrane protein (ICAM-1), prevents it from reaching the medial Golgi. TMD1 or TMD2 alone were accumulated in mitochondria, whereas TMD5 and TMD6 were retained in ER, but were unable to prevent ICAM from reaching the plasma membrane. We conclude that IP 3 R are targeted to the ER membrane only after synthesis of TMDs 1 and 2, and that after co-translational insertion of the remaining TMDs, redundant retention signals present in any pair of TMD retain IP 3 R in the ER.Inositol 1,4,5-trisphosphate receptors (IP 3 R) 1 are intracellular Ca 2ϩ channels that mediate the release of Ca 2ϩ from intracellular stores evoked by receptors that stimulate IP 3 formation (1). Their close relatives, ryanodine receptors (RyR), are another family of intracellular Ca 2ϩ channels with a more restricted distribution and different modes of regulation (2). There are three closely related subtypes (1-3) of mammalian IP 3 R, and although they and their splice variants differ subtly in some aspects of their regulation (3), their structures are likely to be similar (4). All IP 3 R are tetrameric, and each subunit, which may be identical or different within a receptor (3, 5, 6), has a cytosolic IP 3 -binding domain toward the Nterminal (7), separated from a channel toward the C-terminal by a large "modulatory domain" (8). Topology predictions and direct evidence (9, 10) suggest the existence of six transmembrane domains (TMD) toward the C-terminal of each IP 3 R subunit. The last two TMD, together with the intervening loop from each of the four subunits, form the pore (11). Although the transmembrane topology of RyR subunits is unresolved, they are known to have an even number of TMD (probably six or eight) (12) and, as with IP 3 R, the last pair of TMD form the pore (13); both the N and C termini are cytosolic. For both IP 3 R and RyR, the last pair of TMD, together with ϳ25 conserved residues lying ϳ35 residues downstream of them, are also major determinants of assembly of the tetrameric protein (10, 14, 15).Most IP 3 R are expressed in the ER membrane (16 -19), but IP 3 has also been reported to stimulate Ca 2ϩ release from the nuclear envelope (20 -22), nucleoplasmic reticulum (23), Golgi (24), and secretory vesicles (25), although the latter has been challenged (26). IP 3 R are also expressed in the plasma membrane of some cells (2...

Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca 2؉ release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.Ryanodine receptors (RyR) 3 compose a family of intracellular Ca 2ϩ channels that mediate release of Ca 2ϩ from the intracellular stores of excitable and non-excitable cells (1). The three mammalian subtypes of RyR (types 1-3) share ϳ70% amino acid sequence identity, and they are also related to inositol 1,4,5-trisphosphate receptors (IP 3 R) (1, 2). All subtypes of each of the major families of intracellular Ca 2ϩ channels are expressed predominantly, although not exclusively, in the endoplasmic reticulum (ER) or sarcoplasmic reticulum (3, 4). Both families of channels are regulated by diverse intracellular signals, and they also share structural features. All IP 3 R are co-regulated by Ca 2ϩ and inositol 1,4,5-trisphosphate (5), and they are modulated by many additional signals (6). RyR are also regulated by cytosolic Ca 2ϩ and modulated by many of the signals that regulate IP 3 R (1), but RyR subtypes differ in their most important modes of physiological regulation. RyR1, the major isoform of skeletal muscle, is activated by depolarization of the sarcolemma transmitted from dihydropyridine receptors in the plasma membrane to RyR in the junctional sarcoplasmic reticulum (7). In cardiac muscle, the major activator of RyR2 is the local increase in cytosolic [Ca 2ϩ ] that follows depolarizationevoked activation of dihydropyridine receptors. In other cells in which each of the three RyR subtypes can be expressed (8), cytosolic Ca 2ϩ and such signals as cyclic ADP-ribose (9) are probably the major regulators of RyR.Both IP 3 R and RyR form homo-or heterotetrameric assemblies of subunits (3, 10). Each subunit contains a large cytosolic N-terminal domain, a short C-terminal tail, and a stretch of hydrophobic transmembrane domains (TMD), the last two of which form a cation-selective pore with the intervening luminal loop (11-13). The biophysical properties and probably the structure of the pore are similar for...