Interaction between IP₃ receptors and BK channels in arterial smooth muscle: non-canonical IP₃ signaling at work

The dynamic regulation of blood flow in the peripheral circulation comprises multiple mechanisms that work in concert to provide adequate control of blood supply to the various vascular beds that feed organ systems. Blood vessels actively respond to different stimuli to adapt vessel diameter according to the continuously changing metabolic requirements of such organ systems. Among these vasoactive stimuli, shear stress as well as endocrine and paracrine factors play a substantial role in the maintenance of vascular homeostasis and the balance between vasoconstriction and vasodilation by acting on the different cell types that form the vascular wall, such as vascular endothelial and smooth muscle cells (SMCs) (Patterson et al., 2002).

Intracellular calcium concentration ([Ca²⁺]_i) is critical for vascular smooth muscle function, as increased [Ca²⁺]_i triggers SMC contraction. In response to vasoactive ligands that bind to G_q/phospholipase C–coupled receptors, inositol 1,4,5-trisphosphate (IP₃) is produced. IP₃ binds to its receptor (IP₃R) in the membrane of the SMC SR, which facilitates Ca²⁺ release from the SR, raising [Ca²⁺]_i and inducing cell contraction. SMCs can also respond to membrane depolarization via voltage-dependent Ca²⁺ channels (VDCCs) that mediate Ca²⁺ entry from the extracellular milieu independently of SR Ca²⁺ release (Xi et al., 2008; Zhao et al., 2008), triggering vasoconstriction. Moreover, vascular SMCs express plasma membrane large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channels, which are gated by Ca²⁺ with relatively low (micromolar) affinity. As a consequence of a large increase in [Ca²⁺]_i after IP₃-mediated depletion of internal stores, a BK_Ca-dependent hyperpolarizing K⁺ current will induce inactivation of VDCCs, thereby preventing vasoconstriction (Patterson et al., 2002).

The interplay between the different cellular mechanisms that increase [Ca²⁺]_i is important for the control of vascular tone. In this context, the report we review here (Zhao et al., 2010) sheds an interesting light onto how a negative feedback mechanism involving type 1 IP₃ receptors (IP₃R1) and BK_Ca channels is established in cerebral artery SMCs. The findings of Zhao et al. (2010) bring to our attention the possibility that IP₃ may further regulate vascular tone by increasing the apparent Ca²⁺ sensitivity of the BK_Ca channel through a mechanism involving an interaction between the channel and IP₃R1, independently of SR Ca²⁺ release. Using electrophysiological approaches on freshly isolated cerebral artery SMCs, the authors found that IP₃ increased the open probability (P_O) of BK_Ca channels in both cell-attached and excised inside-out membrane patches in a dose-dependent manner. Neither fast Ca²⁺ buffering with BAPTA nor elevated pipette Ca²⁺ concentration (2 mM) altered the ability of IP₃ to increase P_O of BK_Ca channels, suggesting that the observed phenomenon was independent of both intracellular calcium release and plasma membrane calcium influx. Moreover, when the authors examined the Ca²⁺ sensitivity of BK_Ca channels in inside-out patches at a physiological membrane potential of −40 mV, they found that 10 µM IP₃ induced a decrease of the mean apparent dissociation constant (K_d) for Ca²⁺ of the channel from ∼20 to ∼12 µM.

Other reports have suggested that IP₃ may regulate vasoconstriction by inducing a direct coupling between IP₃R1 and canonical transient receptor potential (TRPC) type 3 (TRPC3) channels (Xi et al., 2008; Adebiyi et al., 2010), raising the question of whether IP₃ regulates BK_Ca activity by means of IP₃R1. Indeed, Zhao et al. (2010) found that heparin, an IP₃R blocker as well as a monoclonal antibody against the cytosolic C terminus of IP₃R1, prevented the IP₃-mediated increase in BK_Ca channel P_O. Moreover, experiments performed on cerebral artery SMCs isolated from IP₃R1 knockout mice showed that IP₃ is unable to increase BK_Ca channel P_O in the absence of the IP₃R. This requirement for the IP₃R suggests that the receptor could interact directly with the BK_Ca channel, a hypothesis that was tested by coimmunoprecipitation and immunofluorescence resonance energy transfer (immuno-FRET) studies. Coimmunoprecipitation experiments revealed that both α and β1 subunits of the BK_Ca channel are precipitated by an anti-IP₃R1 antibody, suggesting that the three polypeptides coexist in a higher-order macromolecular complex, whereas immuno-FRET showed that BK_Ca and IP₃R1 localized in close spatial proximity. The data suggest that IP₃ can modulate vascular tone by regulating BK_Ca channel activity independently of the canonical SR Ca²⁺ release pathway, and highlight that this phenomenon requires an interaction between the BK_Ca channel and IP₃R1.

The paper by Zhao et al. (2010) advances our knowledge of the regulation of vascular function by revealing a novel molecular signaling mechanism for IP₃, a ubiquitous second messenger that mediates the actions of many G_q/phospholipase C–coupled vasoconstrictor agonists such as endothelin-1 and angiotensin II (Patterson et al., 2002; Adebiyi et al., 2010). The same group has contributed to the understanding of this non-canonical IP₃ signaling by providing evidence that IP₃Rs can directly couple to plasma membrane ion channels such as TRPC3, thereby modifying their activity (Xi et al., 2008; Adebiyi et al., 2010). In arterial myocytes, both endothelin-1 and IP₃ activate a nonselective cation current (I_Cat) that depends on the functional coupling of TRPC3 and IP₃R1, an interaction that proved to be isoform selective and independent of SR Ca²⁺ release. Although TRPC3-mediated I_Cat would cause membrane depolarization and activation of VDCCs, thereby inducing vasoconstriction, the novel finding that BK_Ca channels are also activated by IP₃ in an IP₃R1-dependent manner raises an interesting question regarding the balance between depolarizing and hyperpolarizing currents in arterial myocytes, which could play a significant role in the control of basal vascular tone and of other active vascular processes such as the myogenic response or endothelium-mediated SMC hyperpolarization. In fact, BK_Ca channels have been implicated in the control of myogenic tone as a hemodynamic autoregulatory mechanism (Hill et al., 2010), and the fact that IP₃ is able to simultaneously regulate TRPC3 and BK_Ca channels provides insight into the role that paracrine factors may play in the modulation of the myogenic response and the local regulation of blood flow. The role of TRPC channels in the control of vasoconstriction has been addressed before, and it has been suggested that the main isoform involved in the myogenic response (i.e., vasoconstriction in response to increased transmural pressure) is TRPC6, whereas TRPC3 would be implicated in the response to vasoconstrictor agents that elevate IP₃ (Welsh et al., 2002; Adebiyi et al., 2010). Beyond local regulation, it will be interesting to probe the importance of this specific coupling between IP₃R1 and BK_Ca channels in the control of blood flow distribution and its role in other vascular beds.

Despite the compelling evidence discussed above, some questions still remain. For instance, all IP₃ stimulations in inside-out patch clamp experiments were performed at concentrations of 10 µM IP₃ or higher, with the support of an IP₃ dose–response curve that yielded maximal stimulations at 10 µM and an apparent K_d for IP₃ of ∼4.1 ± 1.3 µM. Although the authors’ discussion about arterial myocyte global [IP₃]_i is enlightening, we agree that more accurate measurements of basal and agonist-stimulated arterial myocyte [IP₃]_i are required to obtain a clearer picture of the physiologically relevant IP₃ levels that will induce IP₃R1-mediated activation of BK_Ca channels. To this effect, electrophysiological experiments performed at ∼4 µM IP₃ (the apparent K_d indicated by the data) may further clarify the relevance of submaximal IP₃ levels that may be encountered in a physiological context. It was reported previously that Bt-IP₃, a cell-permeable IP₃ analogue, could induce TRPC3-mediated depolarization in endothelium-denuded, SR Ca²⁺-depleted arteries pressurized at 20 mmHg at concentrations as low as 1 µM (Xi et al., 2008), whereas the data provided by Zhao et al. (2010) show no increase in the mean P_O of BK_Ca channels at such IP₃ levels. This discrepancy may be a result of the experimental approach, but we cannot rule out the possibility that the very nature of the interaction between IP₃R1 and plasma membrane ion channels may be different. IP₃Rs have multiple IP₃-binding sites with varying affinities (Foskett et al., 2007), and receptor-activating conformational changes induced by IP₃ binding at different sites could favor interactions with different proteins.

We think that elucidating whether the IP₃-induced shift in Ca²⁺ sensitivity of the BK_Ca channel (from ∼20 to ∼12 µM) is relevant for its biophysical properties, as well as for its physiological role, would be an important step in the clarification of the general picture this paper conveys. However, it remains unsolved whether this effect is in fact a result of a direct molecular interaction between IP₃R1 and the BK_Ca channel. We think that the authors do not provide a conclusive demonstration for the occurrence of a direct interaction, despite that evidence in favor of the requirement of IP₃R1 for IP₃-mediated activation of BK_Ca channels is presented. Heparin, a general IP₃R blocker, and an antibody against IP₃R1 both abolish IP₃-induced BK_Ca activation in inside-out membrane patches. The effect of these IP₃R1-blocking strategies would require an intact and functional receptor (i.e., able to bind IP₃ and activate BK_Ca) excised with the patch. In other work by this group, it was shown that a synthetic peptide corresponding to the N-terminal sequence of the IP₃R containing the TRPC3-binding region activated a Gd³⁺-sensitive, nonselective cation current (I_Cat). Also, membrane-permeant (TAT-conjugated) synthetic peptides were used to probe the coupling between IP₃R1 and TRPC3. Data revealed the presence of a calmodulin- and IP₃R-binding domain in TRPC3 channels that supports the occurrence of the proposed interaction (Adebiyi et al., 2010). To our knowledge, the presence of calmodulin- and IP₃R-binding domains has not been described in BK_Ca channels. The use of these experimental approaches could prove helpful in the validation of the authors’ hypothesis by establishing the actual domains that may be mediating the interaction between IP₃R1 and BK_Ca channels.

IP₃R1 was also shown to localize in close proximity to BK_Ca channels by immuno-FRET. As opposed to the standard FRET approach, which makes use of genetically encoded fluorophores directly coupled to the proteins of interest, immuno-FRET is based on the energy transfer between fluorophores coupled to the secondary antibodies of a standard indirect immunofluorescence. In both methodologies, resonance energy transfer occurs between donor and acceptor molecules located within a specific distance (the Förster radius, R₀) characteristic of each FRET pair, and it is unlikely to occur when the distance between fluorophores is >100 Å (Pietraszewska-Bogiel and Gadella, 2011). Positive immuno-FRET signals are a good indicator of the interaction between IP₃R1 and BK_Ca if the distance between donor and acceptor represents the same separation between these proteins, but this is hard to determine because the three-dimensional structure of the immunocomplex is unknown. Moreover, antibodies are large proteins with a molecular weight of ∼150 kD. Analysis of the crystal structure of various immunoglobulins has shown that the distance between antigen-binding sites can vary between 118 and 185 Å (Saphire et al., 2002). Because R₀ for the FRET pair used by the authors (Cy2/Cy3) is ∼50–60 Å (Zhao et al., 2010), it is possible that the actual distance between IP₃R1 and BK_Ca may be >100 Å, suggesting that energy transfer could be a consequence of the interaction between components of the immunocomplex, regardless of whether or not IP₃R1 and BK_Ca interact and/or localize nearby.

The hypothesis that IP₃R1 and BK_Ca channels are found in close proximity is also supported by coimmunoprecipitation experiments showing that an IP₃R1 antibody is able to pull down both subunits of the BK_Ca channel. Although it is not conclusive evidence of direct interaction, coimmunoprecipitation data clearly suggests the existence of a macromolecular complex that comprises both entities. Despite this attractive inference, Zhao et al. (2010) do not provide a control reverse immunoprecipitation to strengthen their conclusions. Also, probing the presence of molecular chaperones that may stabilize these interactions could provide insight on the trafficking and signaling properties of the IP₃R1–BK_Ca complex, as well as providing explanation for the intracellular normalized FRET data the authors obtained. However, the fact that the regulatory β1 subunit of the channel was coimmunoprecipitated is particularly important; its role as a crucial regulator of channel activity and Ca²⁺ sensitivity (Brenner et al., 2000; Patterson et al., 2002) could provide insight on the mechanisms of BK_Ca modulation by IP₃ and IP₃R1. Experiments aimed at discriminating which BK_Ca subunit mediates the interaction between the channel and the IP₃R or other components of the immunoprecipitated complex will help clarify this issue.

It has been shown previously that TRPC3 and TRPC6 channels can associate with BK_Ca channels in differentiated cells of a podocyte cell line (Kim et al., 2009), whereas recent work shows that, in arterial SMCs, caveolin-1, a lipid raft scaffolding protein, assembles IP₃R1 and TRPC3 channels to signaling complexes (Adebiyi et al., 2011). Moreover, it has been shown that BK_Ca channels are localized to lipid rafts in glioma cells, an interaction that places BK_Ca channels in close proximity to IP₃Rs (Weaver et al., 2007). The involvement of lipid rafts and caveolin-1 in the architecture of these macromolecular signaling complexes brings a bridging element between the different components that constitute them, and paves the road to further inquiry regarding the physiological role of such complexes and the potential interaction with other elements known to localize to plasma membrane microdomains. However, other potential interactions may take place between plasma membrane ion channels and SR membrane components. The STIM/Orai family of proteins has been shown to mediate store-operated Ca²⁺ entry in many different tissues including vascular smooth muscle, and they have been proposed to interact with members of the TRPC channel family to regulate Ca²⁺ entry at cellular domains where the plasma membrane is in close proximity to the SR membrane (Wang et al., 2008). Therefore, BK_Ca channels, IP₃, and IP₃Rs may also be subject to interaction with these ion channel families in the control of SMC Ca²⁺ signals, cell contractility, and vascular tone. Fig. 1 summarizes the potential interactions discussed above.

Figure 1.

IP₃-induced interactions between IP₃Rs and ion channels in arterial SMCs. Induction of Ca²⁺ release from the SR via IP₃R (solid orange arrows) is the best-known mechanism of action of IP₃. Non-canonical IP₃ signaling is independent of SR Ca²⁺ release and involves an IP₃-induced interaction of IP₃R1 with plasma membrane channels such as TRPC3 and BK_Ca (red arrowheads), possibly localized in membrane caveolae. TRPC3 activation by IP₃R1 induces a nonselective cation current (I_Cat; solid green arrow) that depolarizes the cell membrane, activating VDCC and BK_Ca channels (dashed green arrows). BK_Ca channels are also activated by Ca²⁺ (dashed orange arrows). Whereas VDCCs mediate Ca²⁺ influx through the plasma membrane (purple arrow) leading to contraction, K⁺ efflux through BK_Ca channels (blue arrow) leads to hyperpolarization of the cell membrane, which closes VDCCs, leading to SMC relaxation.

The evidence regarding the regulation of vascular homeostasis and, in particular, arterial smooth muscle physiology by non-canonical IP₃ signaling suggests that macromolecular signaling complexes that physically and functionally link the plasma and SR membranes may be an important common theme in terms of the fine modulation of signals that establish vascular SMC excitability and vascular tone, thereby controlling active processes such as the myogenic response. At the same time, they hint at the specific possibility that IP₃-mediated regulation of BK_Ca channels may not only involve the IP₃R but also other molecules present in these signaling microdomains. The recent determination of the crystal structure of the cytoplasmic Ca²⁺-sensing domain of the BK_Ca channels (Wu et al., 2010; Yuan et al., 2010) will certainly provide further insight to this matter by means of structure-driven mutational analysis to elucidate the specific regions of BK_Ca channels that interact with IP₃R1. Careful dissection of their components and integration of their independent and interdependent activities in a physiologically sound model of vascular function will be the next step in the advancement of this field.

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