Perspective

Receptor-Operated Cation Entry—More Than Esoteric Terminology?

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Science's STKE  27 Jul 2004:
Vol. 2004, Issue 243, pp. pe35
DOI: 10.1126/stke.2432004pe35

Abstract

Many hormones and neurotransmitters elicit an increase in the intracellular calcium concentration by binding to phospholipase C–linked G protein–coupled receptors. Activated receptors signal to calcium-permeable cation channels in the plasma membrane, which are distinct from those engaged by emptying of intracellular stores of calcium. The TRPC family of the mammalian homologs of the Drosophila transient receptor potential (TRP) cation channel represents likely molecular correlates underlying receptor-operated cation entry. While all TRPC family members are gated in a phospholipase C–dependent manner, the exact activation mechanism still remains elusive, although lipids such as diacylglycerol and polyunsaturated fatty acids are potential diffusible messengers. Functional TRPC channel complexes in the plasma membrane are thought to be composed of four distinct subunits whose stoichiometry and composition under physiological conditions are still largely unknown. However, recent progress in defining the combinatorial rules of TRPC channel assembly may lead to the identification of TRPC-dependent ion fluxes in living cells. Because of the large number of TRP proteins and their frequently overlapping functional characteristics, the central question is whether TRP proteins are functionally interchangeable or whether unique physiological roles can be ascribed to them. Receptor-operated cation entry is critically involved in the control of airway and vascular smooth muscle tone; hence, TRPC proteins are promising new drug targets.

When excitable and nonexcitable cells are stimulated by many hormones and neurotransmitters, they respond with an elevation of intracellular calcium concentration ([Ca2+]i). The transient rise of [Ca2+]i is nourished both by calcium release from intracellular stores and by cation entry through the plasma membrane. There is mounting experimental evidence that receptors activate cation entry events distinct from those initiated by store-emptying. Receptor-operated cation channels are gated in response to agonist binding to a cell membrane receptor distinct from the channel protein itself. (This is in contrast to ligand-gated ion channels, which are activated by ligand binding directly to the channel subunits.) Over the past several years, a family of mammalian homologs of the Drosophila transient receptor potential (TRP) visual transduction channel has been identified (1, 2). The TRPC subfamily of these channels comprises likely molecular correlates of receptor-operated cation channels. The activation mechanism, regulation, subunit composition, and exact biological role of TRPC proteins thought to mediate receptor-operated cation entry are still poorly understood.

Receptor-operated cation entry plays an eminent physiological role in smooth muscle cells (3). After receptor activation or a rise in intravascular pressure, activation of nonselective cation channels in vascular smooth muscle cells leads to Na+ influx and depolarization, followed by the recruitment of voltage-gated L-type Ca2+ channels mediating the bulk of the Ca2+ influx and smooth muscle contraction (Fig. 1). Voltage-gated Ca2+ channels are key components of the vasopressor response sequence. However, in some blood vessels, norepinephrine-induced vasoconstriction that depends on the presence of external Ca2+ ions can be observed, and agonist-evoked contraction of airway smooth muscle is virtually unaffected under conditions in which voltage-dependent Ca2+ influx is blocked [summarized in (3)]. Most notably, receptor-operated cation channels can be activated by agonist on top of Ca2+ influx elicited by emptying of cellular Ca2+ stores or in the presence of low micromolar concentrations of lanthanides, which block store-operated Ca2+ entry. Thus, receptor-operated cation influx is clearly separable from store-operated Ca2+ entry.

Fig. 1.

Schematic diagram of receptor-operated cation entry in smooth muscle cells. Agonist binding to phospholipase C (PLC)–coupling receptors, such as α1-adrenergic (α1R) or m3-muscarinic (m3R) receptors, leads to inositol trisphosphate (IP3)–mediated release of Ca2+ from internal stores (sarcoplasmic reticulum, SR) and diacylglycerol (DAG)–induced cation entry through TRPC channels. Cation entry depolarizes the cell, thus opening voltage-gated Ca2+ channels (CaV) responsible for the bulk of Ca2+ influx. In smooth muscle cells, depolarization is augmented by Ca2+-activated Cl channels (ClCa). Ca2+-activated K+ channels (BK) hyperpolarize the membrane potential. The cytoplasmic Ca2+ is either sequestered into the SR by resident Ca2+-ATPases (SERCA) or extruded into the extracellular space by plasma membrane Ca2+-ATPases and exchangers (not shown).

Because TRPC6, a member of the canonical TRPC subfamily, is abundant in vascular smooth muscle cells, it represents a likely molecular candidate for the vasoconstrictor-activated, Ca2+-permeable, nonselective cation channel. As characterized in native portal vein myocytes, the TRPC channels are activated by Gq/11-coupled receptors signaling to phospholipase C (PLC) and by diacylglycerol (DAG) independent of protein kinase C (PKC) (4). Upon heterologous expression in CHO-K1 cells, TRPC6 behaves as a receptor-activated, nonselective cation channel that is insensitive to the depletion of internal stores and is activated by DAG (5). A systematic comparison of the α1-adrenoceptor–activated Ca2+-permeable cation channel in rabbit portal vein myocytes (which was found to be receptor- but not store-operated) and the heterologously expressed TRPC6 revealed that their biophysical and pharmacological properties are nearly identical (6). Notably, antisense oligonucleotide–mediated suppression of TRPC6 expression in portal vein myocytes decreased the abundance of the TRPC6 protein and inhibited α1-adrenoceptor–activated cation currents, lending further credence to the notion that TRPC6 has a physiological role as a non–store-operated cation channel.

Although all TRPC family members appear to be subject to a gating mechanism that operates through PLC, three members—TRPC3, 6, and 7—form a structural and functional subgroup that is sensitive to DAG. Although these TRPC proteins are generally classified as the DAG-responsive subfamily, it is still a highly contentious issue as to whether DAG can be regarded as a physiological activator of native channel complexes. As deduced from pharmacological inhibition of DAG lipase and DAG kinase, endogenously generated DAG is sufficient for channel activation (5, 7). Likewise, in portal vein myocytes, blocking DAG metabolism or application of exogenous membrane-permeable DAG analogs gives rise to a nonselective cation current in the absence of receptor agonist, indicating that tonic DAG production is sufficient to gate the cation channels in unstimulated cells (4). However, depending on the readout system—fluorescence imaging or electrophysiology—receptor agonists and DAG do or do not display additive effects on TRPC3 and TRPC6 activity, thus confounding rigorous conclusions as to a role of DAG as a physiological second messenger linking PLC-coupled receptors to TRPC3, 6, and 7 activation (7, 8).

So far, a direct interaction of DAG with TRPC3, 6, or 7 proteins has not been demonstrated. Such a direct contact between the lipid messenger and the channel protein might occur similarly to the interaction of capsaicin with defined intracellular and transmembrane regions of TRPV1 (9). In the absence of a mapped DAG contact site in the channel protein, TRPC3, 6, and 7 activation by C1 domain–containing proteins [such as chimaerins, MUNC13s, RasGRPs (Ras guanine nucleotide releasing proteins) (10), and even DAG kinases] cannot be excluded and remains an enticing possibility that deserves experimental clarification.

TRP proteins form a large and functionally diverse protein channel family and are ubiquitously expressed in both vertebrates and invertebrates. The invertebrate TRPs allow the application of powerful genetic approaches to study the function of these cation channels in vivo. In the nematode Caenorhabditis elegans, for instance, expression of mammalian TRPV4 in neurons of OSM-9–deficient worms restores responses to hypertonicity (11), commensurate with an analogous TRPV4 function in mammals. Mammalian TRPC proteins are the closest relatives of Drosophila TRP. It does not come as a surprise that the role of lipid messengers for the activation of TRP and TRPL in the Drosophila eye remains a highly controversial issue. Flies impaired in DAG kinase activity, which leads to an increase in the local DAG concentration upon receptor stimulation, show enhanced spontaneous currents and light responses consistent with the concept that DAGs or metabolites, such as polyunsaturated fatty acids (PUFAs), gate the cation channels (12). An alternative explanation is based on the role of PUFAs as metabolic uncouplers and the observation that metabolic inhibition activates light-sensitive TRP and TRPL channels (13). However, there is recent evidence that metabolic inhibition primarily impairs DAG kinase activity, consistent with the notion of TRP channel gating by DAG (14).

Finally, one may even pose the heretical question of whether the subsummation of TRPC3, 6, and 7 as "the" DAG-sensitive subfamily is correct at all. Recently, patch-clamp recordings on vomeronasal neuron dendrites provided evidence that TRPC2 may also be a DAG-gated cation channel (15). Along these lines, DAG-activated currents were observed in TRPC5-expressing HEK 293 cells (16). An important methodological difference between these recent findings and other studies on DAG-sensitive TRPCs is that the recent studies used electrophysiology, a highly sensitive technique, whereas the previous studies used fluorescence imaging. Thus, it appears to be worth examining whether all TRPCs in fact respond to DAG, albeit with different potency and efficacy.

Another lipid messenger that has been implicated as a modulator of TRP channel function is phosphatidylinositol-4,5-bisphosphate (PIP2). This phospholipid inhibits Drosophila TRP and TRPL as well as mammalian TRPV1 (2). By contrast, constitutive TRPM7 activity is enhanced upon PIP2 addition and is rapidly inactivated by PIP2 hydrolysis (17). Clearly, further experimentation is required to reach a unifying hypothesis to reconcile the discrepant effects of PIP2 on various TRP channels.

Because the composition of functional TRP channel complexes as the molecular basis of receptor-operated cation channels is largely unknown, it has proven difficult to ascribe receptor-activated cation currents to molecularly defined TRP proteins. Several studies have shown that various TRP channels can assemble as heteromeric complexes that differ in their biophysical properties (18, 19). Systematic analyses of the principles of TRP channel formation offer the conceptual framework to assess the gating mechanism, regulation, and physiological role of distinct TRP proteins in their native environment. So far, two principally different themes have evolved from TRP channel heteromultimerization: On the one hand, heteromultimerization can alter the biophysical properties of channel complexes whose individual members can also be functionally expressed alone (for example, TRPV5/6) (18); on the other hand, heteromultimerization is necessary to measurably transport certain TRP channel subunits to the cell membrane (for example, TRPC1/5, TRPM6/7) (19, 20). In most instances, the daunting task of defining the composition of channel complexes under physiological native conditions still remains to be addressed.

Why are there so many genetically and functionally related TRP proteins? For instance, why do we need several DAG-sensitive TRPC channels? Future experiments, preferably in vivo, will have to address the questions of whether the DAG-sensitive TRPC proteins are functionally redundant and whether unique and indispensable physiological roles can be ascribed to individual DAG-responsive TRPCs. A major drawback for all attempts to define the physiological role of TRPC channels as the molecular correlates for receptor-operated cation entry is the lack of specific channel blockers, which may be overcome by small interfering RNA approaches. Receptor-operated cation entry plays a central role in the physiological control of airway and vascular smooth muscle tone. TRPC proteins as likely molecular correlates may represent attractive new drug targets to reduce smooth muscle tone in pathophysiological states, such as asthma and hypertension, thus propelling receptor-operated cation entry far beyond terminology.

Editor’s Note: This Perspective is part of a series related to an E-Conference held at Science’s STKE. Invited participants were asked to provide Perspectives that summarized the ideas, conclusions, areas of controversy, and challenges for future work that emerged in the online discussion at http://stke.sciencemag.org/cgi/forum-display/stkeforum;14.

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