A volume-regulated chloride current (ICl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for ICl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, pICln and ClC-2 ) have been proposed to be the molecular equivalent of ICl.vol, neither P-glycoprotein nor pICln is thought to be a chloride channel or part thereof, and the properties of expressed ClC-2 channels differ from native ICl.vol. Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of ICl.vol in native cells. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I- > Cl- to Cl- > I- but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels, these results indicate that ClC-3 encodes ICl.vol in many native mammalian cells.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M(2) and M(3), neurokinin receptors NK(1) and NK(3), and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

Regulation of gastrointestinal (GI) motility is intimately coordinated with the modulation of ionic conductance expressed in GI smooth muscle and nonmuscle cells. Interstitial cells of Cajal (ICC) act as pacemaker cells and possess unique ionic conductances that trigger slow wave activity in these cells. The slow wave mechanism is an exclusive feature of ICC: Smooth muscle cells may lack the basic ionic mechanisms necessary to generate or regenerate slow waves. The molecular identification of the components for these conductances provides the foundation for a complete understanding of the ionic basis for GI motility. In addition, this information will provide a basis for the identification or development of therapeutics that might act on these channels. It is much easier to study these conductances and develop blocking drugs in expression systems than in native GI muscle cells. This review focuses on the relationship between ionic currents in native GI smooth muscle cells and ICC and their molecular counterparts.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2؉ -activated K ؉ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the ␣-subunit of the large conductance Large conductance Ca 2ϩ -activated K ϩ (BK Ca ) 1 channels are ubiquitously distributed among tissues and are particularly abundant in smooth muscle (1, 2). The activity of BK Ca channels is regulated by membrane potential, intracellular Ca 2ϩ , and phosphorylation (3, 4). Although BK Ca channels are usually not involved in setting resting potential, they play a key role as a negative feedback mechanism to limit depolarization and contraction (5-7). Activation of BK Ca channels is increased by nitric oxide (NO) and atrial natriuretic peptide, which hyperpolarize the membrane and increase the sensitivity of BK Ca channels to Ca 2ϩ (8 -11). Membrane hyperpolarization closes voltage-dependent Ca 2ϩ channels, reduces Ca 2ϩ influx, and leads to a reduction in intracellular Ca 2ϩ concentration and relaxation (1). NO has been reported to stimulate BK Ca channels directly as well as through stimulation of guanylate cyclase and the subsequent increase in cGMP (12-15). In addition, activation of BK Ca channels plays an important role in NO-induced relaxation of smooth muscle (16 -20). cGMP activates cGMP-dependent protein kinase (PKG), which phosphorylates various cytosolic and membrane proteins that regulate smooth muscle tone either directly or indirectly (21,22). Recent studies in native cells suggest that PKG activates BK Ca channels through phosphorylation of the channel (23). These results are supported by biochemical studies of cloned BK Ca channels, which demonstrate PKG-induced phosphorylation of the channel (24).The primary sequence of BK Ca has been determined using molecular cloning techniques in Drosophila (25) and mammals (26 -28). These studies indicate that BK Ca isoforms belong to the voltage-gated K ϩ (K V ) channel superfamily. The primary sequence of the S1-S6 segment of BK Ca channels is homologous to the corresponding regions in K V channels. The long carboxyl terminus is the region of Ca 2ϩ -sensing (29, 30), and cslo-␣ contains a single high affinity phosphorylation site for PKG at Ser-1072 (3). However additional putative PKG phosphorylation sites have been identified in other splice variants (31). Expression of the slo channel in Xenopus oocytes or mammalian cells gives rise to voltage-gated, Ca 2ϩ -sensitive currents with electrophysiological and pharmacological features similar to those of native BK Ca (32-34). However, although many studies of native cells suggest that BK Ca channel activity is also modulated by various protein kinases (35-38), this property has been difficult to reproduce in cloned channels. Two studies in which slo chann...

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Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing the nonselective cation current I(cat). Na(+) influx through NSCC causes depolarizations and activates voltage-dependent Ca(2+) channels, resulting in contraction. The molecular identity of I(cat) in smooth muscle has not been elucidated; however, products of the transient receptor potential (TRP) genes have characteristics similar to native I(cat). We have determined the levels of TRP transcriptional expression in several murine and canine gastrointestinal and vascular smooth muscles and have analyzed the alternative processing of these transcripts. Of the seven TRP gene family members, transcripts for TRP4, TRP6, and TRP7 were detected in all murine and canine smooth muscle cell preparations. TRP3 was detected only in canine renal artery smooth muscle cells. The full-length cDNAs for TRP4, TRP6, and TRP7, as well as one splice variant of TRP4 and two splice variants of TRP7, were cloned from murine colonic smooth muscle. Quantitative RT-PCR determined the relative amounts of TRP4, TRP6, and TRP7 transcripts, as well as that of the splice variants, in several murine smooth muscles. TRP4 is the most highly expressed, while TRP6 and TRP7 are expressed at a lower level in the same tissues. Splice variants for TRP7, deleted for exons encoding amino acids including transmembrane segment S1, predominated in murine smooth muscles, while the full-length form of the transcript was expressed in canine smooth muscles.