Store Depletion Activated Channels in Pulmonary Artery Smooth Muscle

Little is known yet about the ion channels mediating CCE in pulmonary artery smooth muscle. Store depletion-activated cation currents recorded from rat pulmonary artery myocytes (Fig. ID) have amplitudes of only a few pA in the presence of physiological levels of Ca2+ (27). Store operated currents of similarly small amplitude have been recorded from rabbit and mouse arterial (5, 39) and rabbit venous (1, 20) smooth muscle cells. In contrast, large currents of several hundred pA were reported in cultured human pulmonary artery myocytes (14). This may reflect a phenotypic change from contractile to proliferative cells during culture, as CCE is enhanced during cell proliferation (14). There is general agreement from all these studies that cation-selective channels mediate the currents, although the degree of selectivity for Ca2+ over other cations may vary among different blood vessels. Thus the channel in rabbit portal vein shows a high degree of Ca2+selectivity (1) whereas those in aorta and pulmonary artery discriminate poorly between different cations (27, 39). The store-operated current recorded from pulmonary artery myocytes reverses direction close to 0 mV in physiological conditions and is permeable to Ca2+, Na\ K+, Cs+ and Mn2+ (27). Single channel currents have not yet been reported for store-operated channels (SOCs) in PASMCs, but channels reported for rabbit and mouse aorta and rabbit portal vein have conductances of around 3 pS and a relatively low open probability (1, 39). Once activated by store depletion the channels remain active when membrane patches are excised from the cell, but the channels cannot be activated be SERCA inhibitors applied after patch excision (2, 39). This is the behavior expected for a SOC. Store depletion may not be the only mechanism for activating these channels though, because noradrenaline could activate the same channels in excised outside-out patches via protein kinase C (2). This further complicates the distinction between SOC and ROC.

The pharmacology of store-depletion activated channels in vascular smooth muscle is poorly characterized. In rat pulmonary artery the CPA-induced current is blocked by low micromolar concentrations of Ni2+, Cd2+and SKF96365, but it is resistant to La3+, which is effective only at 100 |xM or higher (27). This pharmacological profile matches the pharmacology of CCE and the CPA-induced contraction reported for rat pulmonary artery in the same study, but is inconsistent with the high La3+ sensitivity reported by others for CCE in the same vessels (32). The reason for this discrepancy is unclear, but the latter study did not measure CCE directly. In general the pharmacology of SOCs is poorly defined. Although a number of drugs are widely used as blockers of SOCs, none of them are selective, especially when used at high concentrations. Thus it is unclear if the SOCs and CCE blocked by millimolar concentrations of Ni2+and La3+ in some studies (14, 39) are the same as channels showing high sensitivity to these blockers.

6. Transient Receptor Potential Channels and Their Relationship to Capacitative Ca2+ Entry

The transient receptor potential (TRP) channel was first identified in Drosophila photoreceptors, where a mutation in the trp gene gives rise to a transient rather than sustained membrane response of the photoreceptor to light. This effect was associated with the loss of permeability, leading to the proposal that the trp gene encodes a Ca2+-permeable ion channel (17). This was supported by strong sequence homology between the genes encoding the TRP protein and voltage-gated channels. Electrophysiological studies subsequently confirmed that both trp and the homologous Drosophila gene, trpl, form Ca2+-permeable non-selective channels in heterologous expression systems.

In the mid 1990s, mammalian homologues of the Drosophila trp gene began to emerge and at least 19 genes are now known to encode human TRP proteins. All ofthese proteins consist of 6 putative membrane-spanning domains, domains 5 and 6 being linked by a short hydrophobic segment predicted to be the pore-forming region, with both the N and C termini located intracellularly (Fig. 2). Based on sequence homology/divergence, we now recognize three major subgroups of mammalian TRP proteins: TRPC, TRPV and TRPM. The TRPV nomenclature originates from the vanilloid receptor, which was the first identified member of the family. Of these channels, only the epithelial TRPV6 protein has been suggested to function as a SOC, although this is disputed. The TRPM nomenclature similarly originates from the first identified member, melastatin; none of these proteins have been implicated as a SOC. Several members of the TRPC family of channels have been suggested at some time to play a role in CCE. Thus, TRPC channels have recently received a great deal of interest in relation to CCE in vascular smooth muscle cells.

7. Properties of Transient Receptor Potential (TRPC) Channels

Seven members (TRPC1-C7) of the TRPC family have been identified. All are able to form cation channels in heterologous expression systems, except the human trpc2, which is a pseudo-gene. Structural homologies within the TRPC family and their relationship to the other mammalian TRP channels are illustrated in Figure 2A. Functional channels are thought to require the co-assembly of four TRPC subunits into a tetrameric complex (Fig. 2C). Table 1 lists some of the characteristic biophysical and pharmacological properties of the homomeric channels, measured from in vitro expression systems.

Human Trpc4 Structure Model Tetramer

Figure 2. Predicted structure of TRPC channels. A: Phylogenetic relationship based on sequence alignment between members of the TRPC family and between the TRPC, TRPV and TRPM families. B: Each subunit is thought to comprise 6 membrane-spanning helices and a putative pore-forming region (P) between the fifth and sixth transmembrane domains. C: A homomeric or heteromeric assembly of four subunits is thought to form the functional channel, with the P regions of all subunits contributing to the pore.

Figure 2. Predicted structure of TRPC channels. A: Phylogenetic relationship based on sequence alignment between members of the TRPC family and between the TRPC, TRPV and TRPM families. B: Each subunit is thought to comprise 6 membrane-spanning helices and a putative pore-forming region (P) between the fifth and sixth transmembrane domains. C: A homomeric or heteromeric assembly of four subunits is thought to form the functional channel, with the P regions of all subunits contributing to the pore.

TRPC proteins can also form heteromeric assemblies consisting ofmore than one subunit type. Clear rules governing the possible interactions within the TRPC family have emerged. TRPC2 does not interact with other TRPC proteins, while TRPC1, TRPC4 and TRPC5 can interact with each other, but not with other members of the family, and TRPC3, TRPC6 and TRPC7 can interact with each other, but not with other members of the family (12, 18). A similar distinction can be drawn in relation to the interactions of TRPC proteins with the Drosophila scaffolding protein INAD (identified from the Inactivation-No-After-Potential Drosophila mutant), which contains protein interaction sites known as PDZ domains and forms the backbone of a macromolecular signaling complex with TRP proteins. INAD can associate with TRPC1, TRPC4 and TRPC5, but not with TRPC3, TRPC6 or TRPC7 (12). The cloning of a human INAD-like protein (29) suggests that comparable complexes may be involved in the regulation of mammalian TRPC channels. In support of this, a PDZ domain in the Na+/H+ exchange regulatory factor (NHERF) binds to TRPC4 and TRPC5 as well as to PLC, suggesting that it could act as a scaffolding protein to bring these signaling molecules together (38). It would be interesting to know if NHERF can interact with other TRPC proteins, or if there are distinct scaffolding proteins for different TRPC complexes. Another distinction can be drawn in relation to diacylglycerol (DAG), which interacts directly with TRPC3 TRPC6 and TRPC7 to cause channel activation, but not with TRPC1, TRPC4 or TRPC5 (19, 28). These properties are all consistent with two functionally distinct subgroups within the TRPC family: the TRPC 1/4/5 subfamily, which are most closely related in terms of evolutionary distance, and the TRPC3/6/7 subfamily. Heteromeric channels formed by interactions within these groups can have properties that are quite distinct from the homomeric channels (23, 35).

Table 1. Properties of Heterologously Expressed TRPC Channels

Y(pS)

PJP*

ICM for La3+ (nM)

Ca2+ Modulation

TRPC1

16'

1

5

inhibition

TRPC 2

n.d.

n.d.

n.d.

n.d.

TRPC 3

66

1.5

50

stimulation

TRPC4

41

1,7

>100

inhibition

TRPC 5

48-63

-10

>100

stimulation

TRPC 6

30

5

4

inhibition

TRPC 7

nd

5

-100

stimulation

'measured in Ca2+-free conditions, y, single channel conductance; PcJPKi, Ca2+ permeability relative to Na+ (Data from Refs, 18, 20, 23, 26, 28, 30, 34, 43).

'measured in Ca2+-free conditions, y, single channel conductance; PcJPKi, Ca2+ permeability relative to Na+ (Data from Refs, 18, 20, 23, 26, 28, 30, 34, 43).

When expressed as homomers, all the TRPC proteins form Ca2+-permeable, non-selective cation channels, although there is wide variation in their singlechannel conductance, Ca2+ permeability relative to Na+, sensitivity to La3+, and modulation by extracellular Ca2+ (Table 1). While TRPC5-TRPC7 discriminate strongly between Ca2+ and Na+ ions, the other TRPC channels display little selectivity for Ca2+ over Na+ or other cations. As commonly found in other Ca2+-permeable channels, Ca2+ passing through the pore can modulate TRPC channel activity. Inhibition of TRPC1 and TRPC4 is apparent at physiological (millimolar) levels of extracellular Ca2+, but Ca2+ has been found to stimulate the activity of TRPC3, TRPC5 and TRPC7 channels. Lanthanides are often used as inhibitors of SOCs but, although La3+ blocks TRPC1 and TRPC3 at low micromolar concentrations, it is at least an order of magnitude less potent on TRPC6 and TRPC7 channels, and rather ineffective on TRPC4 and TRPC5 channels. These properties could all be helpful for the identification of particular TRPC channels underlying Ca2+ entry in vascular cells.

The mechanisms underlying the activation of TRPC channels are still controversial. TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 have all been implicated as SOCs at some time. However, much of the evidence is circumstantial and alternative mechanisms are likely. The evidence for and against a store-dependent mechanism of activation was reviewed recently in several articles (26, 43), so it is not covered here. It is however important to realize how controversial this area is when considering the potential role of TRPCs, acting as SOCs, in HPV. One of the problems may be that TRPC activation has so far been studied primarily in in vitro expression systems, which have generated conflicting results. This may reflect varying levels of protein expression, because expression levels were recently found to profoundly affect the mechanism by which TRPC3 channels are activated (42). Thus at relatively low levels of expression, TRPC3 was activated by store depletion, but at higher levels of expression store depletion was ineffective and the channel required receptor-coupled PLC to open. In general though, it is thought that TRPC3 and TRPC6 are usually activated directly by DAG, and that TRPC 1, TRPC4, TRPC5 are more likely candidates for SOCs. TRPC7 channels have not been widely studied yet, but store depletion was recently shown to activate the human recombinant TRPC7 channel (30), but not the mouse homologue (28). It is therefore still an open question which TRPCs (if any) mediate store-depletion activated Ca2+ influx. The possibility remains that any one of TRPC 1, TRPC3, TRPC4, TRPC5, TRPC 6 or TRPC7 could contribute, either as homomeric channels or, more likely as constituents of a heteromeric channel complex.

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