Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. Following the identification of the founding member of this family, dTRP, which is from a Drosophila mutant with abnormal visual signal transduction (2), mammalian homologues have been cloned and all of them contain a six-transmembrane domain followed by a TRP motif (XWKFXR). Based on homology, they are divided into three subfamilies: TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) (3). Members of the TRPM subfamily have unusually long cytoplasmic tails at both ends of the channel domain, and some of the family members have an enzyme domain in the C-terminal region. Despite their similarities of structure, TRPMs have different ion-conductive properties, activation mechanisms, and putative biological functions. TRPM1 is down-regulated in metastatic melanomas (4). TRPM2 is a Ca 2ϩ -permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (5, 6), and changes in redox status (7). The TRPM2 gene is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holosencephaly (8). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca 2ϩ -permeable channel (9); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca 2ϩ (10). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (11). TRPM7, another bifunctional protein, has kinase activity in addition to its ion channel activity. TRPM7 is regulated by Mg 2ϩ -ATP and/or inositol 1,4,5-disphosphate and is required for cell viability (12-14). TRPM8 is up-regulated in prostate cancer and other malignancies (15). Recently, it has been shown to be a receptor that senses cold stimuli (16,17).Using a bioinformatics approach, we have identified a member of the human TRPM subfamily that we have called hTRPM3, consistent with the unified TRP nomenclature (3). hTRPM3 contains long N and C termini, although it does not contain any additional enzymatic features. hTRPM3 mRNA is expressed primarily in kidney with lower levels in brain, testis, and spinal cord. When expressed in HEK 293 cells, hTRPM3 is co-localized with the plasma membrane and is capable of mediating Ca 2ϩ entry. This hTRPM3-mediated Ca 2ϩ conductance * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)
Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of this study was to determine expression of a Na+ current in human intestinal ICC and to determine the effects of the Na+ current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c-kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A Na+ current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-d-glucamine abolished and QX-314 (500 microM) blocked the Na+ current, but nifedipine and Ni2+ did not. The Na+ current was activated by shear stress. Single-cell RT-PCR detected mRNA for the Na+ alpha-subunit SCN5A in single human intestinal ICC. Lidocaine (200 microm) and QX-314 (500 microM) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive Na+ channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function.
A Na+ current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na+ current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs+ in the pipette to block K+currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na+ current recorded from jejunal smooth muscle cells by 27 ± 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na+current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na+ current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na+ current.
Tetrodotoxin-resistant Na+currents are expressed in a variety of muscle cells including human jejunal circular smooth muscle (HJCSM) cells. The aim of this study was to determine the molecular identity of the pore-forming alpha-subunit of the HJCSM Na+ channel. Degenerate primers identified a cDNA fragment of 1.5 kb with 99% nucleotide homology with human cardiac SCN5A. The identified clone was also amplified from single smooth muscle cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed expression of full-length SCN5A. Laser capture microdissection was used to obtain highly purified populations of HJCSM cells. RT-PCR on the harvested cells showed that SCN5A was present in circular but not in longitudinal muscle. A similar result was obtained using a pan-Na+ channel antibody. The full-length sequence for SCN5A was obtained by combining standard polymerase chain reaction with 5' and 3' rapid amplification of cDNA end techniques. The intestinal SCN5A was nearly identical to the cardiac SCN5A. The data indicate that SCN5A is more widely distributed than previously thought and encodes the pore-forming alpha-subunit of the tetrodotoxin-resistant Na+ current in HJCSM cells.
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