Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.2 is expressed in smooth muscle cells found in all regions of the canine gastrointestinal (GI) tract and in several vascular tissues. Injection of Kv2.2 mRNA into Xenopus oocytes resulted in the expression of a slowly activating K+ current (time to half maximum current, 97 ± 8.6 ms) mediated by 15 pS (symmetrical K+) single channels. The current was inhibited by tetraethylammonium (IC50 = 2.6 mM), 4-aminopyridine (IC50 = 1.5 mM at +20 mV), and quinine (IC50 = 13.7 μM) and was insensitive to charybdotoxin. Low concentrations of quinine (1 μM) were used to preferentially block the slow component of the delayed rectifier current in native colonic myocytes. These data suggest that Kv2.2 may contribute to this current in native GI smooth muscle cells.
To study the contribution of the Na+‐Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]c were evoked by either depolarisation (−70 mV to 0 mV) or by release from the SR by caffeine (10 mm) or flash photolysis of caged InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+‐induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation‐evoked rise in [Ca2+]c. However, although Ca2+ was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation‐evoked Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+‐Ca2+ exchanger activity. Thus inhibition of the Na+‐Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+‐Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+‐Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+‐Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.
Sarcolemma Ca2+ influx, necessary for store refilling, was well maintained, over a wide range (‐70 to + 40 mV) of membrane voltages, in guinea‐pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP3‐evoked Ca2+ transients.
This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca2+ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. ‐70 mV), Ca2+ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage‐gated Ca2+ channels were largely responsible.
Sarcolemma voltage‐gated Ca2+ currents were not activated following store depletion.
Removal of external Ca2+ or the addition of the Ca2+ channel blocker nimodipine (1 μM) inhibited store refilling, as assessed by the magnitude of InsP3‐evoked Ca2+ transients, with little or no change in bulk average cytoplasmic Ca2+ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca2+ gradient.
Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca2+ gradient. Store access to the gradient allows InsP3‐evoked Ca2+ signalling to be maintained over a wide voltage range in colonic smooth muscle.
Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(KACh) could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to beta-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 microM) reversibly activated a Ba(2+)-sensitive and inwardly rectifying K(+) current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.
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