Yijun Ou

SCN5A is expressed in human jejunal circular smooth muscle cells.

Abstract  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 α‐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 α‐subunit of the tetrodotoxin‐resistant Na+current in HJCSM cells.

Species dependent expression of intestinal smooth muscle mechanosensitive sodium channels

Abstract  A mechanosensitive Na+ current carried by Nav1.5 is present in human intestinal circular smooth muscle and contributes to regulation of intestinal motor function. Expression of this channel in different species is unknown. Our aim was to determine if Na+ currents and message for the α subunit of the Na+ channel (SCN5A) are found in circular smooth muscle cells of human, dog, pig, mouse and guinea pig jejunum. Currents were recorded using patch clamp techniques. Message for SCN5A was investigated using laser capture microdissection and reverse transcription polymerase chain reaction (RT‐PCR). Na+ currents were identified consistently in human and dog smooth muscle cells; however, Na+ current was not found in pig (0/20) or guinea pig smooth muscle cells (0/21) and found only one mouse cell (1/21). SCN5A mRNA was found in circular muscle of human, dog, and mouse, but not in pig or guinea pig, and not in mouse longitudinal or mucosal layers. In summary, SCN5A message is expressed in, and Na+ current recorded from, circular muscle layer of human and dog but not from pig and guinea pig. These data show that there are species differences in expression of the SCN5A‐encoded Nav1.5 channel, suggesting species‐specific differences in the electrophysiological response to mechanical and depolarizing stimuli.

The α1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes

T‐type Ca2+ currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca2+ channel α subunit responsible for these currents has not been determined. We established that the α subunit of the α1H Ca2+ channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole‐cell voltage clamp recordings demonstrated that a nifedipine and Cd2+ resistant, mibefradil‐sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the α1H Ca2+ channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the α1H Ca2+ channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the α1H Ca2+ channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the α1H Ca2+ channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.