1. Volume 129, Issue 5, Pages 1504-1517 (November 2005)
Melastatin-Type Transient Receptor Potential Channel 7 Is Required for Intestinal Pacemaking Activity 
Byung Joo Kim, Hyun–Ho Lim, Dong Ki Yang, Jae Yeoul Jun, In Youb Chang, Chul–Seung Park, Insuk So, Peter R. Stanfield, Ki Whan Kim 
Gastroenterology 
Volume 129, Issue 5, Pages (November 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Electrophysiological properties of ICC clusters and expression of TRPM7 protein. (A) Equimolar substitution of transition metal cations for 2 mmol/L extracellular Ca2+. (B) Equimolar substitution of Cs+ and Li+ for 135 mmol/L extracellular Na+. (C) Population data for the manipulations shown in (A) and (B) are expressed as the degree of depolarization. Values are mean ± SEM. (D) RT-PCR detection of TRPM1 (600 base pairs [bp]), TRPM2 (600 bp), TRPM3 (600 bp), TRPM4 (600 bp), TRPM5 (600 bp), TRPM6 (600 bp), TRPM7 (900 bp), and TRPM8 (600 bp). TRPM2, TRPM3, TRPM7, and TRPM8 are expressed in the cultured ICC clusters. (E) TRPM7 proteins are detected at molecular weight 220 kilodaltons (kDa) by Western blotting. Arrow indicates TRPM7 band.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Comparison of TRPM7-like current in single ICCs with expressed TRPM7 current. A single ICC was identified with phycoerythrin-bound anti–c-kit antibody. (A) Representative TRPM7-like currents in ICCs at 0, 1, or 3 mmol/L [Mg2+]i. A voltage ramp from +100 to −100 mV was applied from a holding potential of −60 mV. (B) Current–voltage relationships for expressed TRPM7 at 0, 1, or 3 mmol/L [Mg2+]i. A voltage ramp from +100 to −100 mV was applied from a holding potential of −60 mV. (C) Concentration-dependent inhibition of TRPM7-like current by [Mg2+]i in single ICCs. The estimated median inhibitory concentration value for free Mg2+ was 960 μmol/L. (D) Representative current–voltage relations obtained in ICCs with extracellular solution containing 120 mmol/L N-methyl-d-glucamine-Cl and 20 mmol/L of the chloride salt of the divalent cations indicated. A voltage ramp from +100 to −100 mV was applied from a holding potential of −60 mV.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Spermine inhibited TRPM7-like current reversibly in single ICCs. (A) The complete removal of external divalents increases both inward and outward monovalent current flow. After complete activation of the monovalent current, spermine was applied to cells and showed strong inhibition. (B) Representative current–voltage relations. The holding potential was −60 mV. A voltage ramp was applied from +100 to −100 mV to activate the current.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Pharmacological properties of pacemaking activity in ICC clusters. 2-APB (A), spermine (B), SKF96365 (C), and quinidine (D) inhibited pacemaking activity. (E) Ruthenium red (10 μmol/L) was applied to ICCs. Pacemaking showed no detectable change. (F) Flufenamic acid (50 μmol/L) decreased pacemaking activity.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Expression of TRPM7 protein in mouse small intestine and cultured ICCs. (A) Double labeling of TRPM7-like immunoreactivity (green) and c-kit–like immunoreactivity (red) within the circular muscle layers of mouse small intestine. The mixed color yellow (arrows) indicates the colocalization of both TRPM7-like and c-kit–like immunoreactivity (bars = 50 μm). (B) Double labeling of TRPM7-like immunoreactivity (green) and c-kit–like immunoreactivity (red) at the level of the myenteric plexus of mouse small intestine. Several c-kit–like immunoreactive cell bodies and fibers are colocalized with TRPM7-like immunoreactivity (yellow) (bars = 50 μm). (C) Double labeling with TRPM7 (green) and c-kit (red) antibodies on cultured ICCs. Cultured ICCs show colocalization of TRPM7-like and c-kit−like immunoreactivity (bars = 25 μm).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Effect of RNAi on TRPM7 expressed in HEK-293 cells. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of RNAi effects expressing a tetracycline (tet)-controlled Flag-TRPM7 construct. RNAi effects are shown (R1). C, control vector transfection; R1, RNAiTRPM7-1 transfection; R2, RNAiTRPM7-2 transfection; R3, RNAiTRPM7-3 transfection. Tubulin was used as an internal control. (B) Densitometric analysis of TRPM7 protein expression presented in (A) (n = 5). Values are mean ± SEM. *P < .05. ⇐ indicates a nonspecific band. IB, immunoblotting.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
RNAi effects in ICC clusters. (A) Transfection of single cells (bar = 100 μm). (B) Transfection of clusters (bar = 100 μm). (C) Under current clamp, RNAiCTRL, RNAiTRPM7-2, and RNAiTRPM7-3 show no change in pacemaking activity compared with the original behavior, but RNAiTRPM7-1 decreased pacemaking from 16 ± 2 cycles per minute in the original to 1.6 cycles per minute. (D) Population data for the manipulations are expressed as contraction frequency (n = 6). Values are mean ± SEM. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
RT-PCR detection of TRPM7 in single ICCs. Gel electrophoresis of RT-PCR product from mouse colon (A) and isolated single intestinal myocyte (B) and ICC (C). RT-PCR products were resolved on 2% agarose gel and visualized by ethidium bromide staining. Size markers were used to indicate the size of the experimental fragments (lane 1). (A) RT-PCR detection of glyceraldehyde phosphate dehydrogenase (GAPDH; house keeping; 170 base pairs [bp]), c-kit (ICC marker; 162 bp), myosin heavy chain (MyHC; smooth muscle marker; 234 bp), protein gene product 9.5 (PGP 9.5, a pan-neural marker; 169 bp), and TRPM7 (207 bp) in mRNAs from mouse colon tissue. Left, Plus-RT (Reverse Transcriptase) control; right, Minus-RT control. (B) Single intestinal myocyte shows RT-PCR fragments for MyHC (234 bp) but not for TRPM7. (C) Single ICC shows RT-PCR fragments for both c-kit(162 bp) and TRPM7 mRNA.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions