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.
Hydrogen sulfide (...) is produced endogenously by l-cysteine metabolism. H2S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation ...reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the ... donor NaHS on NaV1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether ... acts on NaV1.5 by redox reactions. Whole cell Na+ currents were recorded in freshly dissociated human jejunum circular myocytes and NaV1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for ... enzymes cystathionine β-synthase and cystathionine ...-lyase from the human jejunum. NaHS increased native Na+ peak currents and shifted the half-point (...) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed NaV1.5 α subunit with ... in the 10... to 10... M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the ...-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of NaV1.5 peak current. These studies show that ... activates the gastrointestinal Na+ channel, and the mechanism of action of ... is partially redox independent. (ProQuest: ... denotes formulae/symbols omitted.)
Hydrogen sulfide (H
2
S) is produced endogenously by l-cysteine metabolism. H
2
S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through ...reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H
2
S donor NaHS on Na
V
1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H
2
S acts on Na
V
1.5 by redox reactions. Whole cell Na
+
currents were recorded in freshly dissociated human jejunum circular myocytes and Na
V
1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H
2
S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na
+
peak currents and shifted the half-point ( V
1/2
) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na
V
1.5 α subunit with EC
50
s in the 10
−4
to 10
−3
M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg
2+
-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na
V
1.5 peak current. These studies show that H
2
S activates the gastrointestinal Na
+
channel, and the mechanism of action of H
2
S is partially redox independent.
T-type ... 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 ... channel α subunit responsible for these currents has not been determined. We established that the α subunit of the ... 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 ... 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 ... 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 ... channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the ... channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the ... channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice. (ProQuest: ... denotes formulae/symbols omitted.)
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