Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca2+ channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca2+ channel in the pathophysiology of smooth muscle dysmotility.
- Ca2+ channel
- c-src kinase
- inactivation facilitation
the excitability of gastrointestinal smooth muscle is strongly coupled to ion-channel activity. Over the last decade, the combination of molecular biology techniques with electrophysiology has resulted in an improved understanding of not only the molecular basis of the biophysical characteristics, but also the structural basis for the regulation of ion-channel function. Neurotransmitter-mediated modulation of the electrical activity, and hence contractility of smooth muscle, can involve either direct effects on the ligand-gated channel where the receptor forms part of the channel complex or via second-messenger pathways that couple receptor activation to voltage-dependent channels.
Almost every known ion channel is influenced by second messengers that include protein kinase(s)-dependent phosphorylation, phospholipid-mediated activation, and/or calcium-based modulation. The diversity in the signaling pathways activated by either G protein-coupled receptors (GPCR) or tyrosine kinase receptors results in the multiplicity of the ionic currents generated. Conversely, different receptors may demonstrate convergence in their signaling pathways affecting the same ion channel. The basis by which protein kinase(s), lipids, and calcium modulate ion channels is becoming increasingly clear based on our understanding of the structural features of the channel protein. Recent studies are beginning to provide a view that not only are ion channels targets for signaling molecules, but they may also participate in controlling intracellular signaling events. This review is largely focused on recent advances in our understanding of the molecular basis for the regulation of the voltage-dependent Ca2+ channel and its relationship to intracellular signaling pathways. The reader is referred to several excellent reviews that define the functional regulation of other types of ionic currents in smooth muscle (2, 13, 20, 35).
Ca2+ SIGNALING AND Ca2+ CHANNELS
Perhaps one of the most ubiquitous signaling molecules that modulates electrical excitability of smooth muscle is the calcium ion. The increase in intracellular Ca2+ involves entry from the extracellular space and/or release from intracellular stores. In addition to its well-known effects on contractility, calcium can affect its own entry through modulation of voltage-dependent and independent ion channels. This type of regulation is important in controlling Ca2+ overload as well as calcium-dependent signaling processes including those affecting gene transcription. For instance, recent studies by Dolmetsch et al. (9) demonstrate that Ca2+ entry through L-type Ca2+ channels provides the basis for activation of cyclic cAMP response element binding protein (CREB) that drives the expression of a number of genes. The activation of CREB occurs as a result of sustained phosphorylation of Ser133 mediated by MAPK. These studies showed that phosphorylation of CREB was attenuated in the presence of the L-type Ca2+ channel blockers demonstrating the requirement for Ca2+ influx. In mice ileal smooth muscle, the translocation of nuclear factor of activated T cells is also dependent on Ca2+ influx through L-type Ca2+ channels (33). The mechanism by which Ca2+-induced signal transduction occurs is now realized as an intricate process that uses specific domains of the Ca2+ channel.
The Ca2+ channel complex is comprised of at least three subunits, the pore-forming α-subunit, which contains the structural determinants for ion selectivity, conductance, and voltage sensing, and the auxillary β- and α2δ-subunits, which confer regulation of the α-subunit. Mammalian α-subunits are encoded by at least 10 different genes. Historically, the calcium channels have been distinguished by alphabetical subscripts based on the pore-forming α-subunit. The smooth muscle dihydropyridine-sensitive L-type Ca2+ channel is derived from the same gene as that originally identified in the heart and initially designated as α1c. Since 2000, a more unified approach has been adopted using numerical identifiers. According to this nomenclature, the classic L-type calcium channels belong to the Cav1.x family, with the α1c denoted as Cav1.2. The Cav1.2 is encoded by least 53 exons with alternative splicing resulting in tissue-specific variants. Many of the differences in the receptor coupling, particularly with regard to the cardiac and smooth muscle, arise as a result of this isoform specificity. The Cav1.2 gene is alternatively spliced, resulting in at least three isoforms known to be present in smooth muscle, although other COOH-terminal splice variants occur in other tissues. The main difference in the isoforms occurs as a result of alternative splicing of the first exon, which encodes the amino terminal. Exons 1a and 1b are the two first exons of the Cav1.2 that encode for longer and shorter isoforms, respectively, and are designated as Cav1.2a and Cav1.2b. The Cav1.2a was first cloned from the rabbit heart and later also identified in the rat aorta (23). The Cav1.2b gene product lacks the initial 30-amino acid segment with an additional difference of 16 amino acids in the NH2 terminus from the Cav1.2a. The Cav1.2b isoform is largely expressed in smooth muscle cells, including the human jejunum (27), and resembles that of the fibroblast and neuronal class of L-type Ca2+ channels. In the mouse colonic smooth muscle, mRNA and protein expression of both isoforms can be detected (18), although the relative abundance of each is not known. Saada et al. (30) recently characterized the promoter region for exon 1b from smooth muscle. On the basis of RNA protection assays, their findings suggest that smooth muscle cells mainly express the short isoform encoded by exon 1b. A third splice variant of Cav1.2b, with a short NH2 terminus, was cloned from the rabbit lung and includes an insert of 25 amino acids in the intracellular loop between domains 1 and II of the α-subunit. These isoform-specific differences in the cytosolic segments are important in the tissue-specific regulation of the channel function. The cytosolic carboxy and the amino termini contain major sites for signal transduction.
SIGNAL TRANSDUCTION THROUGH THE COOH-TERMINAL SEGMENT OF THE Ca2+ CHANNEL
The COOH-terminal region of Cav1.2 is involved in many important processes that regulate channel function and provide feedback to Ca2+ entry (Fig. 1). This includes an inactivation process that limits entry and a facilitation mechanism that results in enhanced Ca2+ influx. Truncation of the distal portion of the COOH terminus results in enhanced currents suggesting the presence of an inhibitory domain (34). In addition to the major site for PKA-mediated phosphorylation (Ser1928) in the COOH terminal (for review, see Ref. 20), there are at least two calmodulin (CaM) binding sites and a proline-rich domain (PRD) that may be important in the tyrosine kinase-mediated regulation of the calcium channel. The calcium binding protein, CaM, appears to play an important role in both Ca2+-mediated inactivation of the channel as well as in the facilitation of Ca2+ entry. Recent studies (32) indicate a link between calcium-CaM interaction and signal transduction necessary for gene transcription.
Inactivation refers to the termination of Ca2+ entry due to the spontaneous closing of the channel pore. In gastrointestinal smooth muscle, membrane depolarization positive to approximately −30 mV results in the opening of the channel pore and influx of Ca2+. There are two major mechanisms by which inactivation of the Ca2+ channel is achieved, i.e., a classic voltage-dependent inactivation and a Ca2+-induced inactivation. In patch-clamp studies of L-type Ca2+ channels from a large number of gastrointestinal smooth muscle cells, maintained depolarization results in current activation that is followed by an inactivation process that is rapid and complete when Ca2+ is the permeating ion. The inactivation is much slower when Ba2+ is used in place of Ca2+. The Ca2+-induced inactivation of the calcium current is an important feature of the Cav1.2, because it provides a physiological feedback mechanism preventing calcium overload during an action potential. This inactivation process also appears to be intimately linked to the activation of CaM kinase and MAPK and is dependent on the mobility of the COOH terminus. Anchoring the cytosolic COOH-terminal tail to the plasma membrane abolishes the activation of CREB-dependent phosphorylation mediated by MAPK as well as Ca2+-dependent inactivation despite the presence of inward Ca2+ currents (22). The molecular basis for the link between Ca2+-induced inactivation and signal transduction has been recently reviewed by Soldatov (32). An essential determinant in this process is the calcium binding protein CaM. The Cav1.2 contains two CaM-binding sites, an “IQ” motif and an upstream CaM-binding domain (CBD) within the COOH terminus. Under resting states, Ca2+-free CaM is tethered to the LA motif of CBD and lies close to the pore region. As Ca2+ enters through the pore on depolarization, it binds to CaM. Ca2+-CaM binding switches to the downstream IQ motif, which has a high affinity for Ca-CaM. It has been proposed that the shift of Ca2+-bound CaM from the LA to the IQ motif induces a collapse around the pore resulting in inactivation of the channel (32). Subsequent hyperpolarization of the membrane returns the COOH-terminal tail to its original resting state. Evidence that the Ca2+-induced inactivation mechanism is linked to activation of MAPK was determined by studies in which mutations were introduced into the IQ domain (9). Mutants of the Ca2+ channel that were lacking the ability to bind to Ca2+-CaM did not demonstrate CREB or Erk phosphorylation, although Ca2+ influx was unaltered.
In addition to downregulation of Ca2+ entry by inactivation mechanisms, Ca2+ entry can also be enhanced by a process referred to as facilitation. Facilitation of the L-type Ca2+ channel was first demonstrated in toad stomach cells (29). When a train of depolarizing pulses was followed by a single pulse, the amplitude of the Ca2+ current was found to be significantly enhanced. This effect was dependent on Ca2+, because it did not occur when Ba2+ was used as the charge carrier and when intracellular Ca2+ concentrations were buffered with BAPTA. The facilitation was blocked by the CaM inhibitory peptide and requires the activation of the CaM protein kinase (CAMK). With the use of a constitutively active Ca2+-CaM-independent CAMK, Dzhura et al. (10) found that CAMK induces a shift in the opening of single Cav1.2 channels into a mode that favors long-duration openings. Similar to Ca2+-channel inactivation, the facilitation process appears to use the CaM-binding IQ segment. Engineered IQ mimetic peptides on dialysis were found to significantly enhance Ca2+-current facilitation (38). The mechanism by which CAMK-mediated phosphorylation enhances Ca2+ currents is unclear at the present time.
REGULATION OF Ca2+ CHANNELS BY c-src KINASE
Recent studies also suggest that smooth muscle Ca2+ channels are modulated by the nonreceptor tyrosine kinase, c-src kinase. Hollenberg (15) initially demonstrated that contraction of gastric smooth muscle by growth factors EGF and PDGF, which activate tyrosine kinase receptors, requires Ca2+ entry. These and other studies (8) have also shown that G protein-coupled agonists such as angiotensin II and acetylcholine induce contractions via a pathway sensitive to tyrosine kinase inhibitors. Western blot analysis with anti-phosphotyrosine antibodies revealed several tyrosyl phosphorylated proteins enhanced by GPCR and PDGF in rabbit colonic smooth muscle (14, 16). If kinase inhibitors attenuate contractions, it can be surmised that tyrosine phosphatase inhibitors should induce contractions by enhancing kinase activity. Indeed, pervanadate was found to produce contractions of gastric muscle (24). Interestingly, these contractions required extracellular Ca2+ and were blocked by the L-type Ca2+ channel blocker nifedipine. The cellular basis for the enhanced Ca2+ influx by receptor activation was demonstrated by patch-clamp studies in single smooth muscle cells. In both vascular and gastrointestinal smooth muscle, structurally unrelated tyrosine kinase inhibitors attenuated calcium currents (16, 36). Several studies in colonic smooth muscle as well as in vascular cells suggest the nonreceptor tyrosine kinase c-src kinase as the modulator of Cav1.2 (for review, see Ref. 7). Hu et al. (16) showed coimmunoprecipitation of the calcium channel with c-src kinase and inhibition of the currents by dialysis of c-src-specific antibody. PDGF-induced enhancement of Ca2+ currents was inhibited by intracellular application of antibodies to c-src kinase and focal adhesion kinase. A convergence between growth factor receptor and GPCR at the level of c-src kinase was suggested in experiments demonstrating enhancement of Ca2+ currents by the muscarinic M2 receptor. Jin et al. (17) found that in rabbit colonic smooth muscle, acetylcholine enhanced Ca2+ currents under conditions whereby the M3 receptor was blocked either by pretreatment with the receptor antagonist 4DAMP or by intracellular dialysis with anti-Gαq antibody. The enhancement of the current was abolished by intracellular dialysis with anti-Gαi antibody or the M2 receptor antagonist methoctramine. Increases in the Ca2+ currents by M2 receptor activation were also abolished in the presence of c-src-specific inhibitors suggesting that GPCR activation through Gαi couples to Ca2+ channels through an src kinase pathway. Similar findings have also recently been observed in rabbit portal vein (6). The mechanism by which src kinase is activated by GPCR is not entirely clear. Several studies have previously suggested that src kinase activation requires Gβγ stimulation (26). However, the Gαi subunit may also directly couple to src (28).
How does c-src kinase regulate Ca2+ channels? Evidence that Ca2+ channels are under basal regulation of c-src kinase comes from 1) the ability of src inhibitors to attenuate currents in the absence of receptor activation as described above, 2) tyrosine phosphatase inhibitors enhance Ca2+ currents (14, 37), and 3) cotransfection of Cav1.2 with the constitutively active v-src in human embryonic kidney cells results in enhanced currents (17). The binding site for src was identified within the COOH-terminal segments by constructing GST-fusion proteins. The COOH-terminal 377-amino acid fusion protein coprecipitated src in pull-down assays of rabbit colonic smooth muscle lysates (17). Bence-Hanulec et al. (4) demonstrated that α1c was tyrosine phosphorylated at residue 2122 in the COOH-terminal segment. Point mutation of this tyrosine (Y2122F) prevented insulin-like growth factor 1-induced enhancement of neuronal L-type Ca2+ currents.
Src kinase comprises two domains that mediate protein-protein interaction, SH2 and SH3, which lie at the back of the kinase domain (39). In the inactive form, the SH2 domain is bound to a phosphotyrosine in the 527 position, and the SH3 domain is bound to the polyproline helix between the SH2 and the kinase domain. Activation of the kinase requires dephosphorylation at the 527 position and autophosphorylation at the 416 position. With the use of an antibody that specifically recognizes phosphorylated 416 kinase, Hu et al. (16) demonstrated the presence of activated src in rabbit colonic smooth muscle under resting conditions, which explains the regulation of the Ca2+ channels by src kinase in unstimulated conditions. The tyrosine phosphorylation site identified in the rat brain α1c (4) is generally absent in Cav1.2 of other species, including the rabbit, although Ca2+ currents are modulated by c-src kinase. This would suggest that there may be other binding sites associated with src activation. Proline-rich regions with a core of PxxP are the preferred interaction sites for the SH3 domain (19). This core is present in the rabbit, rat, and the human Cav1.2 and may be the binding site for src kinase. GST-fusion proteins of the SH3 domain of src bind to the PRD of the distal COOH terminus (12). Truncation of PRD and regions distal to this domain result in enhanced Ca2+ currents (12, 34). Studies by Gerhardstein et al. (12) show that the PRD tethers the inhibitory domain to the main α-subunit. When COOH-terminal fragments containing PRD and the inhibitory domain are dialyzed into cells expressing truncated Ca2+ channels, the currents are attenuated, suggesting that these fragments can associate with the channel (11). Thus cleaving the inhibitory domain is a possible mechanism that would lend itself to autoregulation of the current amplitude. In several tissue types, including smooth muscle, the α1-subunit is detected as a 190- to 200-kDa band on protein isolation, although the full length α1-subunit corresponds to molecular mass of 240 kDa. The difference has been suggested to be due to an endogenous proteolysis of the COOH-terminal 50-kDa segment (12). If the PRD links the inhibitory domain, it may be speculated that binding of the SH3 domain of src to the PRD may induce dissocation of the distal carboxy terminus resulting in enhanced currents. Interaction of the SH3 domain would also result in unfolding of the src protein allowing for the kinase domain to phosphorylate tyrosine residues within the COOH-terminal region.
SIGNAL TRANSDUCTION THROUGH THE NH2-TERMINAL SEGMENT OF Ca2+ CHANNELS
The initial 46 amino acids of the Cav1.2a isoform constitute a major site for PKC modulation of the Ca2+ channels (5). Because this segment is absent in the major smooth muscle isoform Cav1.2b, the direct regulation of the Ca2+ channel by PKC in smooth muscle is unclear. In vascular smooth muscle, several different agonists may enhance Ca2+ currents through a PKC pathway; however, the final mediator could involve c-src kinase (6). In murine colonic myocytes, the PKC inhibitor GF 109203X was found to inhibit basal Ca2+ currents and to abolish substance P-mediated enhancement (3). Whether this involves direct interaction of PKC with the Ca2+ channel is presently not known.
Ca2+ channels in gastrointestinal inflammation.
The amplitude of the Ca2+ currents is markedly attenuated during inflammation. This decrease in currents has been observed in several different models of colonic inflammation (1, 21, 25). In these studies, the Ca2+ current is reduced by almost 70% under basal conditions. The suppression of the Ca2+ currents was associated with decreased protein expression of the α1c-subunit in the dog colon following ethanol/acetic acid-induced inflammation. However, Kinoshita et al. (21) did not observe changes in protein expression in the rat inflamed colon. Similarly, Kang et al. (18) also did not find changes in mRNA or protein expression of Cav1.2a or Cav1.2b in the mouse dextran sulfate sodium (DSS)-induced colitis model, although Ca2+ currents were significantly suppressed. Whether these differences arise as a consequence of inflammatory insult and/or species need to be examined. Liu et al. (25) further demonstrated that acetylcholine-induced enhancement of the currents was also attenuated with inflammation. This downregulation of the receptor-ion channel coupling process may be related to altered expression of G proteins (31). In the murine DSS-induced colitis, Kang et al. (18) observed a decrease in the basal inhibition of the Ca2+ currents by the src kinase inhibitor, suggesting a possible dysregulation of src-mediated modulation of the Ca2+ channels. Future studies aimed at defining the mechanisms associated with the altered function of ion channels under pathophysiological conditions should be helpful in providing new therapeutic targets to treat smooth muscle dysmotility.
Although it has been known for quite some time that ion channels may undergo significant modulation by second messengers, studies are now beginning to define the molecular determinants. It is also becoming increasingly clear that ion channels are not just simple conduits for ion permeation but may also regulate intracellular signaling pathways. Studies to determine how these processes are altered in pathological conditions are an important and an exciting avenue of future research.
The work in the authors' laboratory has been supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46367 and DK-59777.
- Copyright © 2005 the American Physiological Society