Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) 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 Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog's saccular hair cell

Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundle's compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channel's gate as it opens.     

Cyclic nucleotide-gated ion channels in sensory transduction

Cyclic nucleotide-gated (CNG) channels, directly activated by the binding of cyclic nucleotides, were first discovered in retinal rods, cones and olfactory sensory neurons. In the visual and olfactory systems, CNG channels mediate sensory transduction by conducting cationic currents carried primarily by sodium and calcium ions. In olfactory transduction, calcium in combination with calmodulin exerts a negative feedback on CNG channels that is the main molecular mechanism responsible for fast adaptation in olfactory sensory neurons. Six mammalian CNG channel genes are known and some human visual disorders are caused by mutations in retinal rod or cone CNG genes.     

Vertebrate and invertebrate TRPV-like mechanoreceptors

Our senses of touch, hearing, and balance are mediated by mechanosensitive ion channels. In vertebrates, little is known about the molecular composition of these mechanoreceptors, an example of which is the transduction channel of the inner ear's receptor cells, hair cells. Members of the TRP family of ion channels are considered candidates for the vertebrate hair cell's mechanosensitive transduction channel and here we review the evidence for this candidacy. We start by examining the results of genetic screens in invertebrates that identified members of the TRP gene family as core components of mechanoreceptors. In particular, we discuss the Caenorhabditis elegans OSM-9 channel, an invertebrate TRPV channel, and the Drosophila melanogaster TRP channel NOMPC. We then evaluate basic features of TRPV4, a vertebrate member of the TRPV subfamily, which is gated by a variety of physical and chemical stimuli including temperature, osmotic pressure, and ligands. Finally, we compare the characteristics of all discussed mechanoreceptive TRP channels with the biophysical characteristics of hair cell mechanotransduction, speculating about the possible make-up of the elusive inner ear mechanoreceptor.     

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.     

Computational recognition of potassium channel sequences

MOTIVATION: Potassium channels are mainly known for their role in regulating and maintaining the membrane potential. Since this is one of the key mechanisms of signal transduction, malfunction of these potassium channels leads to a wide variety of severe diseases. Thus potassium channels are priority targets of research for new drugs, despite the fact that this protein family is highly variable and closely related to other channels, which makes it very difficult to identify new types of potassium channel sequences. RESULTS: Here we present a new method for identifying potassium channel sequences (PSM, Property Signature Method), which-in contrast to the known methods for protein classification-is directly based on physicochemical properties of amino acids rather than on the amino acids themselves. A signature for the pore region including the selectivity filter has been created, representing the most common physicochemical properties of known potassium channels. This string enables genome-wide screening for sequences with similar features despite a very low degree of amino acid similarity within a protein family.     

Drosophila TRPN(?=?NOMPC) Channel Localizes to the Distal End of Mechanosensory Cilia

Background

A TRPN channel protein is essential for sensory transduction in insect mechanosensory neurons and in vertebrate hair cells. The Drosophila TRPN homolog, NOMPC, is required to generate mechanoreceptor potentials and currents in tactile bristles. NOMPC is also required, together with a TRPV channel, for transduction by chordotonal neurons of the fly''s antennal ear, but the TRPN or TRPV channels have distinct roles in transduction and in regulating active antennal mechanics. The evidence suggests that NOMPC is a primary mechanotransducer channel, but its subcellular location—key for understanding its exact role in transduction—has not yet been established.

Methodology/Principal Findings

Here, by immunostaining, we locate NOMPC at the tips of mechanosensory cilia in both external and chordotonal sensory neurons, as predicted for a mechanotransducer channel. In chordotonal neurons, the TRPN and TRPV channels are respectively segregated into distal and proximal ciliary zones. This zonal separation is demarcated by and requires the ciliary dilation, an intraciliary assembly of intraflagellar transport (IFT) proteins.

Conclusions

Our results provide a strong evidence for NOMPC as a primary transduction channel in Drosophila mechansensory organs. The data also reveals a structural basis for the model of auditory chordotonal transduction in which the TRPN and TRPV channels play sequential roles in generating and amplifying the receptor potential, but have opposing roles in regulating active ciliary motility.     

Modulation of gap junction channels and hemichannels by growth factors

Gap junction hemichannels and cell-cell channels have roles in coordinating numerous cellular processes, due to their permeability to extra and intracellular signaling molecules. Another mechanism of cellular coordination is provided by a vast array of growth factors that interact with relatively selective cell membrane receptors. These receptors can affect cellular transduction pathways, including alteration of intracellular concentration of free Ca(2+) and free radicals and activation of protein kinases or phosphatases. Connexin and pannexin based channels constitute recently described targets of growth factor signal transduction pathways, but little is known regarding the effects of growth factor signaling on pannexin based channels. The effects of growth factors on these two channel types seem to depend on the cell type, cell stage and connexin and pannexin isoform expressed. The functional state of hemichannels and gap junction channels are affected in opposite directions by FGF-1 via protein kinase-dependent mechanisms. These changes are largely explained by channels insertion in or withdrawal from the cell membrane, but changes in open probability might also occur due to changes in phosphorylation and redox state of channel subunits. The functional consequence of variation in cell-cell communication via these membrane channels is implicated in disease as well as normal cellular responses.     

Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO(3)(-)) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca(2+) influx. Using a pharmacological approach, we provide evidence that NO(3)(-) efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO(3)(-) efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

Sigma receptor photolabeling and sigma receptor-mediated modulation of potassium channels in tumor cells.

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (IK). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in IK, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 microM. SKF10047 also inhibited IK with a similar EC50 (14 microM). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5'-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.     

HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.     

Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch-activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd³⁺) concentrations up to 100 times the K_d for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd³⁺, tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.     

Modulation of Ca-activated K channels of human erythrocytes by endogenous protein kinase C

Single IK_Ca channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC_19-31, a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC_19-31 or chelerythrine. Channel activity was not inhibited by the PMA analog 4α-phorbol 12,13-didecanoate (4αPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK_Ca channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK_Ca channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK_Ca channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

The NAADP receptor: new receptors or new regulation?

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent calcium mobilizing messenger yet discovered. Its action has now been reported in a large number of cell types from a diverse array of organisms, and in some cases linked to the transduction of specific cellular stimuli. However, what is controversial is the nature of its target calcium release channel, as well as the subcellular localization of its receptor. Some have proposed that NAADP activates a novel calcium release channel distinct from the two major classes of channels known, the inositol trisphosphate receptors and ryanodine receptors. However, others have suggested that it acts in a novel way to regulate a known calcium release channel, the ryanodine receptor.     

The cyclic nucleotide gated channel of olfactory receptor neurons

Olfactory transduction proceeds through a G-protein coupled cascade that produces the ubiquitous second messenger cyclic AMP. The cyclic AMP causes a chance in membrane potential by acting directly on an ion channel that allows cations to flow into the cell. This ion channel is one of a new family of ion channels that are activated by intracellular cyclic nucleotides. However, even though they are activated by binding a ligand their amino acid structure shows that they share a common ancestry with voltage activated channels, especially voltage dependent Ca²⁺ channels. In olfactory neurons these channels perform a critical role in the transduction of chemical information in the environment into changes in membrane electrical properties that are transmitted to higher order processing centers in the brain.     

A single intracellular cysteine residue is responsible for the activation of the olfactory cyclic nucleotide-gated channel by NO

The activation of cyclic nucleotide-gated (CNG) channels is the final step in olfactory and visual transduction. Previously we have shown that, in addition to their activation by cyclic nucleotides, nitric oxide (NO)-generating compounds can directly open olfactory CNG channels through a redox reaction that results in the S-nitrosylation of a free SH group on a cysteine residue. To identify the target site(s) of NO, we have now mutated the four candidate intracellular cysteine residues Cys-460, Cys-484, Cys-520, and Cys-552 of the rat olfactory rCNG2 (alpha) channel into serine residues. All mutant channels continue to be activated by cyclic nucleotides, but only one of them, the C460S mutant channel, exhibited a total loss of NO sensitivity. This result was further supported by a similar lack of NO sensitivity that we found for a natural mutant of this precise cysteine residue, the Drosophila melanogaster CNG channel. Cys-460 is located in the C-linker region of the channel known to be important in channel gating. Kinetic analyses suggested that at least two of these Cys-460 residues on different channel subunits were involved in the activation by NO. Our results show that one single cysteine residue is responsible for NO sensitivity but that several channel subunits need to be activated for channel opening by NO.     

Mechanosensitive channels in archaea

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree. Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique. Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats. These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels. The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels. This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model. Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles. In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein. Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels. The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.