Sodium current in human jejunal circular smooth muscle cells Adrian N. Holm, Adam Rich, Steven M. Miller, Peter Strege, Yijun Ou, Simon J. Gibbons, Michael G. Sarr, Joseph H. Szurszewski, James L. Rae, Gianrico Farrugia  Gastroenterology  Volume 122, Issue 1, Pages 178-187 (January 2002) DOI: 10.1053/gast.2002.30346 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Distribution of transient inward current recorded from human jejunal circular smooth muscle cells at a holding voltage of −100 mV. In 73 of 100 cells studied, 2 components of the inward current were identified (A), a component with faster kinetics of activation and inactivation and a second component with slower kinetics of activation and inactivation carried by Ca2+ through L-type Ca2+ channels. The current–voltage relationship of the representative currents shown in A is shown in D, with maximal peak inward current for the 2 components at −30 mV and −10 mV. In 8 of 100 cells, only the faster of the 2 components could be determined definitively. A representative example is shown in B, with initial inward current seen at −60 mV and maximal peak inward current at −30 mV (E). In 19 of 100 cells, only the slower Ca2+ current could be determined definitively. A representative example is shown in C, with initial inward current seen at −50 mV and maximal peak inward current at 0 mV (F). The insets show current traces at −30 mV (solid line) and 0 mV (dashed line) from each of the 3 cells. The outward current seen at positive voltages (e.g., B) is caused by a nonselective cation current activated at voltages positive to 0 mV. Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of removal of Na+ from the bath. (A) Representative current traces obtained using the pulse protocol shown in the inset. Both fast and slower components were present in this cell. (B) Effect of substitution of Na+ with NMDG on inward currents. (C) Current–voltage relationships; the inset shows the mean maximal peak currents for both the fast and slow components in normal Ringer (■) and in NMDG (2). Removal of Na+ resulted in complete loss of the fast component (Na+), with no effect on the slow (Ca2+) current (inset, *P < 0.0001). (D) Normalized mean current–voltage relationship obtained from human jejunal circular smooth muscle cells. Currents were recorded either from cells with no discernible Ca2+ current (n = 8), in the presence of manganese replacing Ca2+ (n = 5), or in the presence of nifedipine (1 μmol/L, n = 9). Inward Na+ current was first noted at approximately −60 mV, and maximal peak inward current was seen at −25 mV. Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Steady-state activation and inactivation of the Na+ current obtained using the pulse protocols shown in the insets. Fits were obtained from the average V½ and slope (k) calculated for each experiment (n = 8 for the activation experiments, n = 7 for the inactivation experiments). V½ for activation was −47 mV, and k was 4.8. V½ for inactivation was −78 mV, and k was −3.2. A window current was present at voltages from −75 mV to −60 mV. Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Internal and external QX314 blocks the Na+ current. (A) Block of the Na+ current by internal QX314 (100 μm, ○, n = 4). The data were fit with a single-order exponential. In contrast, there was no decrease in Na+ current in control records obtained in the absence of QX314 (▵, n = 3). (B) Effect of external QX314 (500 μm, n = 4). External QX314 also blocked the Na+ current. Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Slowing of inactivation by ATX II. (A) Representative recording of Na+ current obtained from a single smooth muscle using the pulse protocol shown in the inset. The cell was held at −100 mV. (B) Records from the same cell 10 minutes after addition of ATX II (10 μmol/L). Inactivation was slowed by ATX II. (C) Results of exponential fits to the inactivation curves (n = 5). Control data were best fit with a single exponential with a τ of 4.8 ± 0.4 milliseconds at −35 mV (▿). In contrast, the data obtained in the presence of ATX II were best fit with a double exponential with τ values of 3.9 ± 0.8 milliseconds and 50 ± 19 milliseconds at −35 mV (○, 2). Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 SCN5A is present in human jejunal smooth muscle cell libraries. (A) cDNA bands obtained using PCR with primers designed against portions of the published sequence of SCN5A. (B) The bands were of the expected size for SCN5A, and sequencing of the bands (2 with overlapping sequence) showed 99% homology with the published SCN5A sequence. (C) cDNA bands obtained using RT-PCR from messenger RNA isolated from human jejunal circular smooth muscle cells collected by laser-capture microdissection. Bands were the expected size for SCN5A and glyceraldehyde-3-phosphate dehydrogenase (phosphorylating; control), and the products were confirmed by sequencing. No band was seen using c-kit primers. Gastroenterology 2002 122, 178-187DOI: (10.1053/gast.2002.30346) Copyright © 2002 American Gastroenterological Association Terms and Conditions