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.     

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.     

Functional co-assembly among subunits of cyclic-nucleotide-activated, nonselective cation channels, and across species from nematode to human.

Cyclic-nucleotide-activated, nonselective cation channels have a central role in sensory transduction. They are most likely tetramers, composed of two subunits (alpha and beta or 1 and 2), with the former, but not the latter, being able to form homomeric cyclic-nucleotide-activated channels. Identified members of this channel family now include, in vertebrates, the rod and cone channels mediating visual transduction and the channel mediating olfactory transduction, each apparently with distinct alpha- and beta-subunits. Homologous channels have also been identified in Drosophila melanogaster and Caenorhabditis elegans. By co-expressing any combination of two alpha-subunits, or alpha- and beta-subunits, of this channel family in HEK 293 cells, we have found that they can all co-assemble functionally with each other, including those from fly and nematode. This finding suggests that the subunit members so far identified form a remarkably homogeneous and conserved group, functionally and evolutionarily, with no subfamilies yet identified. The ability to cross-assemble allows these subunits to potentially generate a diversity of heteromeric channels, each with properties specifically suited to a particular cellular function.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

KATP channels gated by intracellular nucleotides and phospholipids.

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir6. 1 or Kir6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4, 5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications.     

The TRP ion channel family

Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.     

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.     

Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels

Native ion channels are precisely tuned to their physiological role in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising multiple types of subunits. Cyclic nucleotide-gated (CNG) channels of olfactory neurons are tetramers and require three types of subunits, CNGA2, CNGA4, and CNGB1b, to exhibit properties necessary for olfactory transduction. Using fluorescently tagged subunits and fluorescence resonance energy transfer (FRET), we find the subunit composition of heteromeric olfactory channels in the surface membrane is fixed, with 2:1:1 CNGA2:CNGA4:CNGB1b. Furthermore, when expressed individually with CNGA2, CNGA4 and CNGB1b subunits were still present in only a single copy and, when expressed alone, did not self-assemble. These results suggest that the precise assembly of heteromeric olfactory channels results from a mechanism where CNGA4 and CNGB1b subunits have a high affinity for CNGA2 but not for self-assembly, precluding more than one CNGA4 or CNGB1b subunit in the channel complex.     

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.     

MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.     

Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.     

Distinct pools of epithelial sodium channels are expressed at the plasma membrane

The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (alpha, beta, and gamma). Channel maturation within the biosynthetic pathway involves cleavage of the alpha and gamma subunits by furin and processing of N-linked glycans on alpha, beta, and gamma to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.     

Coordination of membrane excitability through a GIRK1 signaling complex in the atria

Control of heart rate is a complex process that integrates the function of multiple G protein-coupled receptors and ion channels. Among them, the G protein-regulated inwardly rectifying K+ (GIRK or KACh) channels of sinoatrial node and atria play a major role in beat-to-beat regulation of the heart rate. The atrial KACh channels are heterotetrameric proteins that consist of two pore-forming subunits, GIRK1 and GIRK4. Following m2-muscarinic acetylcholine receptor (M2R) stimulation, KACh channel activation is conferred by the direct binding of G protein betagamma subunits (Gbetagamma) to the channel. Here we show that atrial KACh channels are assembled in a signaling complex with Gbetagamma, G protein-coupled receptor kinase, cyclic adenosine monophosphate-dependent protein kinase, two protein phosphatases, PP1 and PP2A, receptor for activated C kinase 1, and actin. This complex would enable the KACh channels to rapidly integrate beta-adrenergic and M2R signaling in the membrane, and it provides insight into general principles governing spatial integration of different transduction pathways. Furthermore, the same complex might recruit protein kinase C (PKC) to the KACh channel following alpha-adrenergic receptor stimulation. Our electro-physiological recordings from single atrial KACh channels revealed a potent inhibition of Gbetagamma-induced channel activity by PKC, thus validating the physiological significance of the observed complex as interconnecting site where signaling molecules congregate to execute a coordinated control of membrane excitability.     

The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.

Cation channels in the DEG/ENaC family are proposed to detect cutaneous stimuli in mammals. We localized one such channel, DRASIC, in several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and/or acid-evoked nociception. Disrupting the mouse DRASIC gene altered sensory transduction in specific and distinct ways. Loss of DRASIC increased the sensitivity of mechanoreceptors detecting light touch, but it reduced the sensitivity of a mechanoreceptor responding to noxious pinch and decreased the response of acid- and noxious heat-sensitive nociceptors. The data suggest that DRASIC subunits participate in heteromultimeric channel complexes in sensory neurons. Moreover, in different cellular contexts, DRASIC may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.     

Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission

Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.     

Functional analysis of cloned opioid receptors in transfected cell lines

Opioids modulate numerous central and peripheral processes including pain perception, neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (μ, δ, κ). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein α subunits and/or βγ subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K⁺ channels and reduces adenylyl cyclase and Ca²⁺ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca²⁺ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca²⁺ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH₃ cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.     

The bacterial K+ channel structure and its implications for neuronal channels.

Voltage-gated K+ (Kv) channels play a central role in generating action potentials and rhythmic patterns, as well as in dendritic signal processing in neurons. Recently, the first structure of a member of the K+ channel family was solved. Although this channel is from bacteria and has a streamlined body plan with no voltage gating, it establishes the architecture of the functional core of the voltage-gated (K+) channels and their relatives. This architecture explains the crucial features of ion permeation and blockade, and gives some strong hints about gating. The bacterial K+ channel structure is the central piece in a puzzle; it remains to be seen how it will fit together with other domains of the Kv channels, with auxiliary subunits, and with other signal transduction molecules.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

A specific subset of transient receptor potential vanilloid-type channel subunits in Caenorhabditis elegans endocrine cells function as mixed heteromers to promote neurotransmitter release

Transient receptor potential (TRP) channel subunits form homotetramers that function in sensory transduction. Heteromeric channels also form, but their physiological subunit compositions and functions are largely unknown. We found a dominant-negative mutant of the C. elegans TRPV (vanilloid-type) subunit OCR-2 that apparently incorporates into and inactivates OCR-2 homomers as well as heteromers with the TRPV subunits OCR-1 and -4, resulting in a premature egg-laying defect. This defect is reproduced by knocking out all three OCR genes, but not by any single knockout. Thus a mixture of redundant heteromeric channels prevents premature egg laying. These channels, as well as the G-protein G alpha(o), function in neuroendocrine cells to promote release of neurotransmitters that block egg laying until eggs filling the uterus deform the neuroendocrine cells. The TRPV channel OSM-9, previously suggested to be an obligate heteromeric partner of OCR-2 in sensory neurons, is expressed in the neuroendocrine cells but has no detectable role in egg laying. Our results identify a specific set of heteromeric TRPV channels that redundantly regulate neuroendocrine function and show that a subunit combination that functions in sensory neurons is also present in neuroendocrine cells but has no detectable function in these cells.