Melastatin-Type Transient Receptor Potential Channel 7 Is Required for Intestinal Pacemaking Activity
2005; Elsevier BV; Volume: 129; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.08.016
ISSN1528-0012
AutoresByung Joo Kim, Hyun–Ho Lim, Dong Ki Yang, Jae Yeoul Jun, In Youb Chang, Chul‐Seung Park, Insuk So, Peter Stanfield, Ki Whan Kim,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoBackground & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut. Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut. Interstitial cells of Cajal (ICCs) are the pacemakers in gastrointestinal (GI) muscles and also mediate or transduce inputs from the enteric nervous system. Because of the central role of ICCs in GI motility, loss of these cells would be extremely detrimental. Research into the biology of ICCs provides exciting new opportunities to understand the etiology of diseases that have long eluded comprehension. Discovering the molecules involved in the generation of pacemaker activity in ICCs may lead to dramatic new therapies for chronic GI diseases that result in lifelong suffering. ICCs generate the electrical pacemaker activity (slow wave) in GI muscles.1Ward S.M. Burns A.J. Torihashi S. Sanders K.M. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.J Physiol. 1994; 480: 91-97Crossref PubMed Scopus (765) Google Scholar, 2Huizinga J.D. Thuneberg L. Kluppel M. Malysz J. Mikkelsen H.B. Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.Nature. 1995; 373: 347-349Crossref PubMed Scopus (1278) Google Scholar, 3Sanders K.M. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.Gastroenterology. 1996; 111: 492-515Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar Slow waves propagate within ICC networks, conduct into smooth muscle cells via gap junctions, and initiate phasic contractions by activating Ca2+ entry through L-type Ca2+ channels. The pacemaker activity in the murine small intestine is due mainly to periodic activation of nonselective cation channels (NSCCs).4Koh S.D. Jun J.Y. Kim T.W. Sanders K.M. A Ca2+-inhibited non-selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal.J Physiol. 2002; 540: 803-814Crossref PubMed Scopus (140) Google Scholar Transient receptor potential (TRP) channels were first cloned from Drosophila species (TRP and transient receptor potential like protein [TRPL]) and constitute a superfamily of proteins that encode a diverse group of Ca2+-permeable NSCCs.5Clapham D.E. TRP channels as cellular sensors.Nature. 2003; 426: 517-524Crossref PubMed Scopus (2218) Google Scholar The TRP family is divided into 3 subfamilies: classic (TRPC), vanilloid type (TRPV), and melastatin type (TRPM).5Clapham D.E. TRP channels as cellular sensors.Nature. 2003; 426: 517-524Crossref PubMed Scopus (2218) Google Scholar TRPC channels mediate cation entry in response to phospholipase C activation, whereas TRPV proteins respond to physical and chemical stimuli, such as changes of temperature, pH, and mechanical stress.5Clapham D.E. TRP channels as cellular sensors.Nature. 2003; 426: 517-524Crossref PubMed Scopus (2218) Google Scholar The 8 TRPM family members differ significantly from other TRP channels in terms of domain structure, cation selectivity, and activation mechanisms.5Clapham D.E. TRP channels as cellular sensors.Nature. 2003; 426: 517-524Crossref PubMed Scopus (2218) Google Scholar The defecation rhythm in both vertebrate animals and Caenorhabditis elegans requires inositol 1,4,5-triphosphate receptors.6Suzuki H. Takano H. Yamamoto Y. Komuro T. Saito M. Kato K. Mikoshiba K. Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor.J Physiol. 2000; 525: 105-111Crossref PubMed Scopus (164) Google Scholar Inositol 1,4,5-triphosphate receptors in C elegans are encoded by a single gene, itr1. Genetic studies have shown that itr1 (gene for IP3 receptor) is required for the ultradian rhythm underlying defecation and also for ovulation.7Dal Santo P. Logan M.A. Chisholm A.D. Jorgensen E.M. The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans.Cell. 1999; 98: 757-767Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar In C elegans, the members of the TRPM subfamily are GON-2, Gon-2–like proteins (GTL-1 and 2), and CED-11. GTL-1 is involved in the regulation of the defecation rhythm generator, most likely as a part of a calcium signaling network.8Vriens J. Owsianik G. Voets T. Droogmans G. Nilius B. Invertebrate TRP proteins as functional models for mammalian channels.Pflugers Arch. 2004; 449: 213-226PubMed Google Scholar GTL-1 shows higher homology to TRPM7 (33.3%), TRPM1 (35.0%), and TRPM3 (34.0%) than to other TRPM members.8Vriens J. Owsianik G. Voets T. Droogmans G. Nilius B. Invertebrate TRP proteins as functional models for mammalian channels.Pflugers Arch. 2004; 449: 213-226PubMed Google Scholar Among these 3 members of TRPM subfamily, TRPM7 has been well studied by other researchers and was chosen as a candidate for intestinal pacemaking activity. Initially, TRPC4 was also suggested as a molecular candidate for the calcium-inhibited NSCC responsible for pacemaking.2Huizinga J.D. Thuneberg L. Kluppel M. Malysz J. Mikkelsen H.B. Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.Nature. 1995; 373: 347-349Crossref PubMed Scopus (1278) Google Scholar, 9Torihashi S. Fujimoto T. Trost C. Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal requirement of calcium influx and localization of TRP4 in caveolae.J Biol Chem. 2002; 277: 19191-19197Crossref PubMed Scopus (180) Google Scholar, 10Walker R.L. Koh S.D. Sergeant G.P. Sanders K.M. Horowitz B. TRPC4 current have properties similar to the pacemaker current in interstitial cells of Cajal.Am J Physiol Cell Physiol. 2002; 283: 1637-1645Crossref Scopus (97) Google Scholar However, TRPC4, when expressed, was activated by stimulating G protein–coupled receptors rather than by store depletion.11Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gundermann T. Schultz G. Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.J Biol Chem. 2000; 275: 17517-17526Crossref PubMed Scopus (364) Google Scholar, 12Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. TRPC1 and TRPC5 form a novel cation channel in mammalian brain.Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 13Plant T.D. Schaefer M. TRPC4 and TRPC5 receptor-operated Ca2+-permeable nonselective cation channels.Cell Calcium. 2003; 33: 441-450Crossref PubMed Scopus (137) Google Scholar, 14Zhu M.H. Lee Y.M. Jin N.G. So I. Kim K.W. The transient receptor potential protein homologue TRPC4/5 as a candidate for the nonselective cationic channel activated by muscarinic stimulation in the murine stomach.Neurophysiology. 2003; 35: 330-335Crossref Scopus (12) Google Scholar Further, in our study, slow waves could be recorded even in TRPC4 knockout mice.15Lee K.P. So I. Kim K.W. TRPC4 might not be involved in the generation of slow waves.Biophys J. 2003; 84: 106aAbstract Full Text Full Text PDF Scopus (49) Google Scholar TRPV6 was originally reported as a calcium-inhibited NSCC or calcium release–activated calcium channel.16Yue L. Peng J.B. Hediger M.A. Clapham D.E. CaT1 manifests the pore properties of the calcium-release-activated calcium channel.Nature. 2001; 410: 705-709Crossref PubMed Scopus (322) Google Scholar However, ruthenium red, a blocker of TRPV6, had no effect on pacemaking activity in our preliminary study. In C elegans, there are 2 types of calcium conductance, store independent (TRPM7-like) or store operated, and both contribute to the defecation rhythm.17Estevez A.Y. Roberts R.K. Strange K. Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells.J Gen Physiol. 2003; 122: 207-223Crossref PubMed Scopus (34) Google Scholar Thus, TRPC4 and TRPV6, as candidates for the calcium-inhibited or store-operated calcium channel, might play a role in pacemaking activity, but would do this together with a TRPM7-like store-independent calcium conductance. In this study, we have tried to find a TRPM7-like store-independent calcium conductance and have used electrophysiological, molecular biological, and immunohistochemical techniques to establish the close relationship between NSCC in ICCs and a mammalian TRP homologue, TRPM7. BALB/c mice (8–13 days old) of either sex were anesthetized with ether and killed by cervical dislocation. The small intestines from 1 cm below the pyloric ring to the cecum were removed and opened along the mesenteric border. Luminal contents were washed away with Krebs–Ringer bicarbonate solution. The tissues were pinned to the base of a Sylgard dish, and the mucosa was removed by sharp dissection. Small tissue strips of intestinal muscle (consisting of both circular and longitudinal muscles) were equilibrated in Ca2+-free Hanks solution (in mmol/L: KCl 5.36, NaCl 125, NaOH 0.34, Na2HCO3 0.44, glucose 10, sucrose 2.9, and HEPES 11) for 30 minutes. The cells were then dispersed with an enzyme solution containing collagenase (Worthington Biochemical Co, Lakewood, NJ) 1.3 mg/mL, bovine serum albumin (Sigma Chemical Co, St Louis, MO) 2 mg/mL, trypsin inhibitor (Sigma) 2 mg/mL, and adenosine triphosphate 0.27 mg/mL. Cells were plated onto sterile glass coverslips coated with murine collagen (2.5 μg/mL; Falcon/BD, Franklin Lakes, NJ) in a 35-mm culture dish. The cells were subsequently cultured at 37°C in a 95% oxygen/5% carbon dioxide incubator in a smooth muscle growth medium (Clonetics Corp, San Diego, CA) supplemented with 2% antibiotics/antimycotics (Gibco, Grand Island, NY) and murine stem cell factor (5 ng/mL; Sigma). All experiments on single cells were performed on cells cultured for 1 day. ICCs were identified immunologically with anti–c-kit antibody (phycoerythrin-conjugated rat anti-mouse c-kit monoclonal antibody; eBioscience, San Diego, CA) at a dilution of 1:50 for 20 minutes. The physiological salt solution used to bathe cells (Na+-Tyrode) in cultured ICC clusters contained (mmol/L) KCl 5, NaCl 135, CaCl2 2, glucose 10, MgCl2 1.2, and HEPES 10, adjusted to pH 7.4 with NaOH. The pipette solution contained (mmol/L) KCl 140, MgCl2 5, K2 adenosine triphosphate 2.7, NaGTP 0.1, creatine phosphate disodium 2.5, HEPES 5, and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid 0.1, adjusted to pH 7.2 with KOH. In case of single ICCs, the cells were bathed in a solution containing (mmol/L) KCl 2.8, NaCl 145, CaCl2 2, glucose 10, MgCl2 1.2, and HEPES 10, adjusted to pH 7.4 with NaOH. The pipette solution contained (mmol/L) Cs-glutamate 145, NaCl 8, Cs-2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid 10, and HEPES-CsOH 10, adjusted to pH 7.2 with CsOH. The whole-cell configuration of the patch-clamp technique was used to record membrane currents (voltage clamp) or potentials (current clamp) from cultured ICCs. An Axopatch I-D (Axon Instruments, Foster City, CA) was used to amplify membrane currents and potentials. The command pulse was applied with an IBM-compatible personal computer and pClamp software (version 6.1; Axon Instruments). The data were filtered at 5 kHz and displayed on an oscilloscope, a computer monitor, and a pen recorder (Gould 2200; Gould, Valley View, OH). Results were analyzed by using pClamp and Origin software (Microcal Origin version 6.0). All experiments using ICCs were performed at 30°C. Human embryonic kidney (HEK)-293 cells transfected with the Flag-murine LTRPC7/pCDNA4-TO construct were grown on glass coverslips with Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, blasticidin (5 μg/mL), and zeocin (0.4 mg/mL). TRPM7 (LTRPC7) expression was induced by adding 1 μg/mL tetracycline to the culture medium. Whole-cell patch-clamp experiments were performed at 21°C–25°C 24 hours after induction by using cells grown on glass coverslips. The solutions were the same as those used for recording whole-cell currents in single ICCs. Total RNA was extracted by using an RNeasy Mini Kit (Qiagen, Inc), and reverse transcription of total RNA was performed by using random hexamer primers and Superscript II-RT (Life Technologies Inc), according to the manufacturer's instructions. Polymerase chain reaction (PCR) primers are as follows: the first PCR amplification with upstream primers (TRPM1-OF, 5′-TGTTAGACCAGTCTTACA-3′ for TRPM1; TRPM2-OF, 5′-AGCCAGAAAGTGGGGAAG-3′ for TRPM2; TRPM3-OF, 5′-AGCTCCTGGACCAGTCCT-3′ for TRPM3; TRPM4-OF, 5′-TAGGCTGCCGGCTGACCC-3′ for TRPM4; TRPM5-OF, 5′-TCAACTTCGGAGGGTCTG-3′ for TRPM5; TRPM6-OF, 5′-GTGAACCTCCACCGCTTC-3′ for TRPM6; TRPM7-OF, 5′-TCTGTGAGTACCCCATCC-3′ for TRPM7; and TRPM8-OF, 5′-TTCCGGCTCCACTCTTCT-3′ for TRPM8) and downstream primers (TRPM1-OR, 5′-CTGTGTGGTTTTTCACTT-3′ for TRPM1; TRPM2-OR, 5′-GCCATTGTTGATGGCATT-3′ for TRPM2; TRPM3-OR, 5′-ATCTCTCTCATCTTCTCT-3′ for TRPM3; TRPM4-OR, 5′-TCCTTACAGAGACAGACA-3′ for TRPM4; TRPM5-OR, 5′-GGGTGTTGGGGTCACCAT-3′ for TRPM5; TRPM6-OR, 5′-GTCATCCACCATGTTGCT-3′ for TRPM6; TRPM7-OR, 5′-CCCTGCTGAATGGCAGTA-3′ for TRPM7; and TRPM8-OR, 5′-CTTCACCACCATGTAGAA-3′ for TRPM8) was performed for 40 cycles under the following conditions: denaturing at 94°C for 2 minutes, annealing at 50°C for 1 minute, and polymerization at 72°C for 1 minute. Nested PCR amplifications with primers (for TRPM1: TRPM1-IF, 5′-AGCACGATGAGCAGGTTG-3′ and TRPM1-IR, 5′-TAAGCTCCTGTTCTGCCA-3′; for TRPM2: TRPM2-IF, 5′-TATGTCCGGGTCTCCCAG-3′ and TRPM2-IR, 5′-GTAGATTGTATGCAGAGT-3′; for TRPM3: TRPM3-IF, 5′-ACAAACAGGATGAGCAGC-3′ and TRPM3-IR, 5′-ATTCCCAGAGTGAAAATG-3′; for TRPM4: TRPM4-IF, 5′-CTGGGCTCTTTGACTTGG-3′ and TRPM4-IR, 5′-CATGACAGATGATGATGA-3′; for TRPM5: TRPM5-IF, 5′-GGAAGAAGCGAGGCAAGT-3′ and TRPM5-IR, 5′-TGACCAACAGGCAAAGGA-3′; for TRPM6: TRPM6-IF, 5′-CTCACCATCCCTCGGCTG-3′ and TRPM6-IR, 5′-CTCCTTGGCTTCCCGGGC-3′; for TRPM7: TRPM7-IF, 5′-CAGCCAAGTTGCAAAAGC-3′ and TRPM7-IR, 5′-CAACAGGAAAACTTCAAG-3′; and for TRPM8: TRPM8-IF, 5′-AATAAAAGCTCGTTGTAC-3′ and TRPM8-IR, 5′-ATAAGCGAAGACAACGAA-3′) were performed for 40 cycles under the following conditions: denaturing at 94°C for 2 minutes, annealing at 50°C for 1 minute, and polymerization at 72°C for 1 minute. The PCR products, predicted as 600 base pairs (TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, and TRPM8) and 900 base pairs (TRPM7), were separated on 1.5% agarose gel by electrophoresis. The identification of the PCR products was confirmed by DNA sequencing. To confirm that the ICC has TRPM7 messenger RNA (mRNA), we performed single-cell reverse-transcription PCR (RT-PCR). Each single cell was collected by applying negative pressure to a cell in contact with a recording pipette, lifting the cell out of the bath, and immediately expelling the cell from the pipette into a PCR tube, which contained lysis buffer. A complementary DNA library constructed from mouse colon was used as a positive control. RT-PCR was performed with 40 cycles at an annealing temperature of 60°C. The following PCR primers were used to detect cell type–specific mRNA species: c-kit (Y00864, ICC) sense, nucleotides 2706–2726 and antisense, nucleotides 2847–2867; smooth muscle myosin heavy chain (NM_013607; smooth muscle) sense, nucleotides 5721–5745 and antisense, nucleotides 5930–5954; protein gene product 9.5 (a pan-neuronal marker; AF172334) sense, nucleotides 22–44 and antisense, nucleotides 171–190; and TRPM7 sense, TCTGTGAGTACCCCATCCCAGCCAAGT and antisense, TCCAGCAGCACCCACATGTTTCAGAGT. All primers were obtained from Keystone Labs (Camarillo, CA) or Bioneer (Korea). In some cells, PCR was performed with 40 cycles twice at an annealing temperature of 60°C. The amplified products were separated by electrophoresis on a 2% agarose/1× Tris, acetic acid, and ethylenediaminetetraacetic acid gel, and the DNA bands were visualized by ethidium bromide staining. Nonspecific amplification and spurious primer–dimer fragments were controlled by omitting the template from the PCR amplification. Cultured ICCs and whole-mount preparations from small intestine of BALB/c mice were used for immunohistochemistry. Animal welfare protocols approved by the animal center of Seoul National University were followed. For whole-mount preparations, the mucosa was removed by sharp dissection, and the remaining muscle layer was stretched before fixation. Both cultured ICCs and whole-mount preparations were fixed in cold acetone (4°C) for 5 minutes. After fixation, they were washed in phosphate-buffered saline (PBS; 0.01 mol/L; pH 7.4) and immersed in 0.3% Triton X-100 in PBS. After blocking with 1% bovine serum albumin (Sigma) in 0.01 mol/L PBS for 1 hour at room temperature, they were incubated with a rat monoclonal antibody raised against c-kit (Ack2; eBioscience) at 0.5 μg/mL or goat polyclonal antibody against TRPM7 (Abcam, Cambridgeshire, UK) in PBS for 24 hours (4°C). After a rinse in PBS at 4°C, they were labeled with the fluorescein isothiocyanate–coupled donkey anti-goat immunoglobulin G secondary antibody (1:100; Jackson Immunoresearch Laboratories, Baltimore, MD) or Texas red–conjugated donkey anti-rat immunoglobulin G (1:100; Jackson Immunoresearch Laboratories) for 1 hour at room temperature. Control tissues and sections were prepared by omitting either primary or secondary antibodies from the incubation solutions. For double immunostaining, specimens were incubated with a mixture of antibody raised against TRPM7 and antibody raised against c-kit for 24 hours at 4°C. After a thorough wash with PBS, the mixture of labeled secondary antibodies was incubated for 1 hour at room temperature. Tissues were examined by using a laser scanning confocal microscope (FV 300; Olympus, Japan) with an excitation wavelength appropriate for fluorescein isothiocyanate (495 nm) or Texas red (590 nm). Final images were constructed with Flow-View software (Olympus). Western blotting was performed by using lysates of 107 HEK-293 cells and ICCs. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using 6% polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and analyzed by anti-FLAG and TRPM7 antibodies. All procedures used standard methods. Cultured ICC clusters were loaded with the acetoxymethyl ester form of fura-2 (5 μmol/L; diluted from 1 mmol/L stock in dimethyl sulfoxide) in normal medium for 25 minutes at 37°C. The recording of [Ca2+]i and of other intracellular divalent cations was performed with a microfluorometric system consisting of an inverted fluorescence microscope (Diaphot 300; Nikon, Japan) with a dry-type fluorescence objective lens (40×; numerical aperture 0.85), a photomultiplier tube (type R 1527; Hamamatsu, Japan), and a PTI-Deltascan illuminator (Photon Technology International Inc). Cells were superfused at a flow rate of 2 mL/min. Light was provided by a 75-W xenon lamp (Ushino, Japan). To control excitation frequency, a chopper wheel alternated the light path to monochromators (340 and 380 nm) with a frequency of 5 or 10 Hz. A short-pass dichroic mirror passed emission light of <570 nm onto the photomultiplier tube, and intensity was measured at 510 nm. A mechanical image mask was placed in the emission path to limit measurement to a single cell. Both data acquisition and control of light application were performed with computer software (Felix version 1.1; Photon Technology International Inc, Brunswick, NJ). Because of uncertainties in calibrating the fura-2 signals in intact cells, no attempt was made to calibrate [Ca2+]i or other intracellular divalent cation concentrations; instead, all results are reported as changes in the 340 nm/380 nm signal ratio. The RNA interference (RNAi) against TRPM7 corresponded to coding regions 205–225, 1845–1865, and 4087–4107, relative to the first nucleotide of the start codon of murine TRPM7 (GenBank accession number NM_021450). These RNAis are termed RNAiTRPM7-1, RNAiTRPM7-2, and RNAiTRPM7-3, respectively. The RNAiCTRL was a commercially available psiRNA-hH1GFPzeo vector (catalog no. ksirna3-gz11; InvivoGen Inc). Relative intensities of protein bands were analyzed with a GS-710 Image Densitometer (Bio-Rad Laboratories, Hercules, CA). Data are expressed as means ± SEM. Differences between the data were evaluated by Student t test. A P value <.05 was taken to indicate a statistically significant difference. The n values reported in the text refer to the number of cells used in the patch-clamp experiments. Under current clamp, cells in cultured ICC clusters had a mean resting membrane potential of −58 ± 3 mV and produced electrical pacemaking activity (n = 136). The frequency of this pacemaking was 16 ± 2 cycles per minute (n = 136) at 30°C. To compare the conductance sequence of divalent cations, we first examined the effect of external divalent cations on pacemaking activity. We replaced external 2 mmol/L Ca2+ with equimolar Ba2+, Mn2+, Sr2+, Zn2+, Ni2+, or Co2+ and recorded the membrane potential. All these divalent cations induced depolarization. The degree of depolarization was by 43 ± 4 mV with Zn2+ (n = 5), 31 ± 3 mV with Ni2+ (n = 6), 26 ± 2 mV with Ba2+ (n = 10), 21 ± 3 mV with Co2+ (n = 5), 12 ± 2 mV with Mn2+ (n = 12), and 8 ± 2 mV with Sr2+ (n = 12; Figure 1A). Thus, cations depolarized in the following sequence: Zn2+ > Ni2+ > Ba2+ > Co2+ > Mn2+ > Sr2+ > Ca2+ (Figure 1C). There are 2 possible mechanisms for this depolarization: an increase of inward current or a decrease of outward current. To assess whether these divalent cations permeate and so increase the pacemaking NSCC, we performed ratiometric calcium imaging. In each case, we found an increase in the F340/F380 ratio. The change in ratio indicates an increased intracellular divalent cation concentration even in the absence of external calcium; this suggests that these cations do indeed carry inward current. In contrast, in the presence of external Ca2+, the divalent cations induced hyperpolarization, acting as blockers of pacemaking activity. These results suggest that these transition metal ions can carry NSCC in murine small intestine instead of Ca2+ but block this current when extracellular Ca2+ is present. It has recently been suggested that Erg K+ channels are involved in pacemaking activity,18Zhu Y. Golden C.M. Ye J. Wang X.Y. Akbarali H.I. Huizinga J.D. ERG K+ currents regulate pacemaker activity in ICC.Am J Physiol Gastrointest Liver Physiol. 2003; 285: G1249-G1258PubMed Google Scholar and divalent cations are known to inhibit Herg K+ channels expressed in xenopus oocytes.19Ho W.K. Kim I. Lee C.O. Earm Y.E. Voltage-dependent blockade of HERG channels expressed in Xenopus oocytes by external Ca2+ and Mg2+.J Physiol. 1998; 507: 631-638Crossref PubMed Scopus (40) Google Scholar However, the order of potency in which divalent cations inhibit Herg is different from that associated with their ability to cause depolarization in cultured ICC clusters. To investigate the permeability of monovalent cations, we replaced external 135 mmol/L Na+ with an equimolar concentration of Cs+ or Li+ and recorded the membrane potential. The degree of depolarization was 32 ± 2 mV with Cs+ (n = 5) and 6 ± 1 mV with Li+ (n = 4; Figure 1B), thus indicating a sequence of Cs+ > Li+ > Na+ (Figure 1C). The results suggest that Cs+ and Li+ carry NSCC better than does Na+. With the results concerning depolarization to hand, we searched the characteristics of divalent cation permeability sequences among TRP channels. The results we found in cultured ICC clusters were compatible with the divalent cation conductance sequence of TRPM7.20Monteilh-Zoller M.K. Hermosura M.C. Nadler M.J. Scharenberg A.M. Penner R. Fleig A. TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions.J Gen Physiol. 2003; 121: 49-60Crossref PubMed Scopus (458) Google Scholar In contrast, in TRPV5, the divalent cation permeability sequence was Ca2+ > Ba2+ > Sr2+ > Mn2+; in TRPV6, it was Ca2+ > Sr2+ ≈ Ba2+ > Mn2+21Hoenderop J.G. Vennekens R. Muller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. Function and expression of the epithelial Ca2+ channel family comparison of mammalian ECaC1 and 2.J Physiol. 2001; 537: 747-761Crossref PubMed Scopus (235) Google Scholar; and in TRPC6, it was Ca2+ > Ba2+ > Sr2+. Thus, the cation selectivity is that expected of TRPM7. To reinforce the candidacy of TRPM7, both TRPM7 mRNA (Figure 1D) and protein (Figure 1E) were detected in the cultured ICC clusters by RT-PCR and Western blotting techniques, respectively. Thus, TRPM7 exists in the cultured ICC clusters. In addition, TRPM2, TRPM3, and TRPM8 mRNAs were also present in cultured ICC clusters. To record the NSCC involved in pacemaking activity, we performed whole-cell voltage-clamp recordings in cultured single ICCs. Single ICCs were identified with phycoerythrin-bound anti–c-kit antibody. A voltage ramp from +100 mV to −100 mV evoked an outward-rectifying cation current at positive potentials with standard bath solution and with pipette solutions lacking magnesium adenosine triphosphate (n = 12; Figure 2A). These features are very similar to those associated with the recently cloned TRPM7 channel.22Nadler M.J. Hermosura M.C. Inabe K. Perraud A.L. Zhu Q. Stokes A.J. Kurosaki T. Kinet J.P. Penner R. Scharenberg A.M. Fleig A. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.Nature. 2001; 411: 590-595Crossref PubMed Scopus (831) Google Scholar The typical feature of TRPM7 is its inhibition by internal Mg2+ or magnesium adenosine triphosphate. The TRPM7-like currents in single ICCs were recorded under various internal magnesium concentrations. The amplitude of TRPM7-like current was indeed larger with lower than with higher internal magnesium ion concentration [Mg2+]i. The median inhibitory concentration was 960 μmol/L (Figure 2C). Similar results were obtained in HEK cells expressing TRPM7 (Figure 2B). Next we tested the conductance of the TRPM7-like current to Ca2+ and other divalent cations. Significant inward currents were measured with all the divalent cations tested as the sole charge carrier in the extracellular solution. At a concentration of 20 mmol/L, the rank order determined from the inward current amplitude at −100 mV was Ni2+ > Ba2+ > Mn2+ > Ca2+ (n = 7; Figure 2D). When normalized to the inward current amplitude in 20 mmol/L Ca2+, we obtained relative values of 2, 1.875, and 1.125 for Ni2+, Ba2+, and Mn2+, respectively. After the current had stabilized in normal bath solution, a divalent-free solution was perfused into the bath, and this resulted in large inward and outward currents with little rectification (n = 5; Figure 3A and B). TRPM7 has been shown to be blocked by external polyvalent cations such as spermine.23Kerschbaum H.H. Kozak J.A. Cahalan M.D. Polyvalent cations as permeant probes of MIC and TRPM7 pores.Biophys J. 2003; 84: 2293-2305Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Perfusing spermine (100 μmol/L) into the bath in divalent-free solution reduced both inward current and outward current. The block was reduced by washing out the spermine for 2–3 minutes before returning it to the normal bath solution. The block was voltage dependent, with an average block
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