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The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.
1. Complementary DNAs for the ATP-gated ion channel subunits P2X1 (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole-cell patch clamp method. The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (PX/PNa) were 2.3, 1.0, 1.0, 0.95, 0.72, 0.5, 0.29, 0.16, 0.04 and 0.03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2-methylethanolamine, tris(hydroxymethyl)-aminomethane, tetraethylammonium and N-methyl-D-glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mM calcium outside, 154 mM sodium inside), PCa/PNa values were 3.9 and 2.2, respectively (corrected for ionic activities). 5. ATP-evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0.3-30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP-evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.
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There is little information about the mechanisms by which G-protein-coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPgammaS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the K(ACh) channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245-253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BK(Ca) channel). Open probability (P(O)) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca(2+) and Galphao-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when P(O) was measured over 5-s intervals. This was related to the variable frequency of openings (as in the K(ACh) channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as P(O) increased 525-fold in three stages.
Patch fluorometry has emerged as a new approach to the study of the structure-function relationship in membrane-embedded functional ion channels. Simultaneous fluorescent and electrical recordings are achieved from a small number of channels in a cell-free membrane patch, yielding high recording sensitivities. Further improvement of this approach should permit direct observation of the gating motion of a single-channel protein.
Channel-forming colicins are bacterial toxins that spontaneously insert into the inner cell membrane of sensitive bacteria to form voltage-gated ion channels. It has been shown that the channel current and the conformational flexibility of colicin E1 channel domain depend on the membrane surface potential, which is regulated by the anionic lipid content and the ion concentration. To better understand the dependence of colicin structure and dynamics on the membrane surface potential, we have used solid-state NMR to investigate the topology and segmental motion of the closed state of colicin Ia channel-forming domain in membranes of different anionic lipid contents and ion concentrations. Colicin Ia channel domain was reconstituted into membranes with different POPG and KCl concentrations. 1H spin diffusion experiments indicate that the protein contains a small domain that inserts into the hydrophobic center of the 70% anionic membrane, similar to when it binds to the 25% anionic membrane. Measurements of C-H and N-H dipolar couplings indicate that, on the sub-microsecond time scale, the protein has the least segmental mobility under the high-salt and low-anionic lipid condition, which has the most physiological membrane surface potential. Measurement of millisecond time scale motions yielded similar results. These suggest that optimal channel activity requires the protein to have sufficient segmental rigidity so that entire helices can undergo cooperative conformational motions that are required for translocating the channel-forming helices across the lipid bilayer upon voltage activation.
Voltage-gated Na+ channels are composed of an alpha subunit of 260 kDa associated with beta subunits of 33-36 kDa. Alpha subunits have four homologous domains (I to IV) containing six transmembrane alpha helices (S1-S6). The S4 segments serve as voltage sensors and move outward to initiate activation. The S5 and S6 segments and the short membrane-associated loops between them form the pore. Fast inactivation is mediated by closure of an inactivation gate formed by the intracellular loop between domains III and IV. The 3-D structure of the inactivation gate has been determined bv NMR spectroscopy, revealing the conformation of the pore-blocking IFM motif. Peptide scorpion toxins that alter gating of Na+ channels bind to the extracellular ends of the IIS4 and IVS4 segments, trap them in either an activated or non-activated position, and thereby selectively alter channel activation or inactivation. Voltage sensor-trapping may be a general mechanism of toxin action on voltage-gated ion channels. Local anaesthetics block the pore of Na+ channels by binding to a receptor site in segment S6 in domains III and IV. Anticonvulsants and antiarrhythmic drugs also interact with this site. A high-affinity Na+ channel blocker has recently been developed with this site as its target. The emerging knowledge of the molecular mechanisms of Na+ channel gating and drug block may allow development of novel therapeutics for epilepsy, cardiac arrhythmia and persistent pain syndromes.
Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.
Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca2+ and Na+ channels were analyzed by electrophysiological techniques (using Ba2+ as charge carrier through Ca2+ channels) and by binding studies with specific ligands. With NGF, Na+ current (I(Na)) density increased in parallel with neurite extension. Ba2+ current (I(Ba)) density and Ca2+ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I(Na) density. With forskolin, neurite outgrowth was linked to an apparent increase in I(Ba) density similar to the one induced by NGF, while no change in I(Na) was observed. Dihydropyridine-sensitive (L-type) as well as omega-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I(Ba), binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca2+ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca2+ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics.
Cannabinoids are a structurally diverse group of mostly lipophilic molecules that bind to cannabinoid receptors. In fact, endogenous cannabinoids (endocannabinoids) are a class of signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. They are synthesized from lipid precursors in plasma membranes via Ca(2+) or G-protein-dependent processes and exhibit cannabinoid-like actions by binding to cannabinoid receptors. However, endocannabinoids can produce effects that are not mediated by these receptors. In pharmacologically relevant concentrations, endocannabinoids modulate the functional properties of voltage-gated ion channels including Ca(2+) channels, Na(+) channels, various types of K(+) channels, and ligand-gated ion channels such as serotonin type 3, nicotinic acetylcholine, and glycine receptors. In addition, modulatory effects of endocannabinoids on other ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions and transporters for neurotransmitters have also been demonstrated. Furthermore, functional properties of G-protein-coupled receptors for different types of neurotransmitters and neuropeptides are altered by direct actions of endocannabinoids. Although the mechanisms of these effects are currently not clear, it is likely that these direct actions of endocannabinoids are due to their lipophilic structures. These findings indicate that additional molecular targets for endocannabinoids exist and that these targets may represent novel sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems. This review focuses on the results of recent studies indicating that beyond their receptor-mediated effects, endocannabinoids alter the functions of ion channels and other integral membrane proteins directly.
cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.
Total colourblindness (OMIM 216900), also referred to as rod monochromacy (RM) or complete achromatopsia, is a rare, autosomal recessive inherited and congenital disorder characterized by photophobia, reduced visual acuity, nystagmus and the complete inability to discriminate between colours. Electroretinographic recordings show that in RM, rod photoreceptor function is normal, whereas cone photoreceptor responses are absent. The locus for RM has been mapped to chromosome 2q11 (ref. 2), however the gene underlying RM has not yet been identified. Recently, a suitable candidate gene, CNGA3, encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel, a key component of the phototransduction pathway, has been cloned and assigned to human chromosome 2q11 (refs 3,4). We report the identification of missense mutations in CNGA3 in five families with RM. Homozygous mutations are present in two families, whereas the remaining families show compound heterozygous mutations. In all cases, the segregation pattern of the mutations is consistent with the autosomal recessive inheritance of the disease and all mutations affect amino acids that are highly conserved among cyclic nucleotide gated channels (CNG) in various species. This is the first report of a colour vision disorder caused by defects other than mutations in the cone pigment genes, and implies at least in this instance a common genetic basis for phototransduction in the three different cone photoreceptors of the human retina.
Cyclic nucleotide-gated cation channels, which are permeable to monovalent and divalent cations, are expressed in a number of tissues. cDNAs encoding cGMP-gated cation channel subunits have been cloned in retinal rods, cones, olfactory neuroepithelium, pineal gland, aorta, testis, heart, and most recently kidney. Patch clamp studies have identified and characterized cGMP-gated cation channels in the cortical collecting duct (CCD) and inner medullary collecting duct (IMCD). cGMP-gated cation channels in kidney share many biophysical and molecular properties with the retinal rod cGMP-gated channel. However, unlike the retinal rod channel, the cGMP-gated cation channel in kidney is inhibited by cGMP and stimulated by increased calcium levels. In the IMCD the cGMP-gated cation channel mediates electrogenic sodium absorption which is inhibited by ANP via cGMP. Recently, cGMP-gated cation channel poly(A+) RNA has been identified in other nephron segments by RT-PCR and in situ PCR hybridization. Furthermore, cGMP-gated cation channel protein has also been identified in all nephron segments by Western blot analysis. These observations suggest that cGMP-gated cation channels, or closely related gene products, may play an important physiological role in all nephron segments. Hormones that increase intracellular cGMP may regulate sodium, and perhaps calcium, uptake in nephron segments proximal to the IMCD. Increases in cell sodium and calcium may regulate other transport and signaling pathways.
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Stimulation of cultured cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c-fos induction. For this increase in TRE-binding activity, Ca2+ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca2+ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of 45Ca2+ uptake and TRE-binding activity by NMDA or KA suggested that Ca2+ influx not only triggered sequential activation of Ca(2+)-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca2+ influx through voltage-gated Ca2+ channels, whereas stimulation of NMDA receptors caused Ca2+ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca(2+)-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.
Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a "cutoff" is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the alpha subunit. We now demonstrate that these residues in the glycine alpha1 and the gamma-aminobutyric acid rho1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine alpha1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the gamma-aminobutyric acid rho1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.
Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5'-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5'-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50-80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction.
We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.