I. INTRODUCTION

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Anion channels in the heart have been the subject of electrophysiological examination for nearly four decades dating back to the original work in 1961 of Hutter and Noble (188) and Carmeliet (43). In the 1970s, there was general agreement that an increase in Cl conductance was largely responsible for the initial rapid phase of repolarization of the action potential of cardiac Purkinje fibers. However, later studies raised serious doubts about the identity of this Cl conductance, and the eventual application of the patch-clamp technique to enzymatically dispersed cardiac cells in the early 1980s relegated Cl channels in the heart, like in some other tissues, to a minor and mundane role of membrane "leak." In 1989, though, the demonstration that a time- and voltage-independent anion leak conductance was tightly linked to regulation by the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway (13, 164) provided new impetus for further studies of Cl channels in the heart.

During the past decade, an ever-increasing amount of energy has been devoted to the functional and molecular characterization of anion channels as well as transport and exchange proteins in sarcolemmal and internal membranes of cardiac cells and to efforts to reveal their physiological and possible pathophysiological role. A representation of our present understanding of the different types of anion channels as well as transport and exchange proteins found in cardiac sarcolemmal and internal membranes, and some of their intracellular signaling pathways, is illustrated schematically in Figure 1. Initially, six different types of sarcolemmal Cl currents were functionally identified in cardiac cells. These included Cl currents regulated by the adenylyl cyclase-cAMP-PKA pathway (I_Cl.PKA), protein kinase C (PKC) (I_Cl.PKC), cell volume (I_Cl.vol), cytoplasmic Ca²⁺ (I_Cl.Ca), purinergic receptors (I_Cl.ATP) (see Ref. 2 for review), and a basally active Cl current (I_Cl.b). This list of putative sarcolemmal anion channels has been simplified somewhat by new evidence that suggests that I_Cl.PKA, I_Cl.PKC, and I_Cl.ATP in heart may all be mediated by a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel and evidence that I_Cl.b and I_Cl.vol may be generated by the same protein. Molecular candidates responsible for I_Cl.vol and I_Cl.Ca presently include the ClC-3 and CLCA1 gene products, and there is emerging evidence for expression of a new type of sarcolemmal anion channel in some cardiac cells, which generates an inwardly rectifying Cl current (I_Cl.ir) and may be encoded by ClC-2.

View larger version (70K): [in this window] [in a new window]   Fig. 1. Schematic representation of cardiac anion channels, transport and exchange proteins, and their intracellular signaling pathways. Anion channels and transport or exchange proteins are indicated in yellow, and their corresponding molecular entities or candidates (?) are indicated in parentheses. ICl.PKA, Cl current regulated by adenylyl cyclase-cAMP-protein kinase A pathway; CFTR, cystic fibrosis transmembrane conductance regulator; M1-6, CFTR transmembrane spanning segments 1-6; M7-12, CFTR transmembrane spanning segments 7-12; NBDA, nucleotide binding domain A; NBDB, nucleotide binding domain B; R, regulatory subunit; P, phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC); PP, serine-threonine protein phosphatases; 1a-AR, -adrenergic receptor type 1a; G?, unidentified heterotrimeric G protein; ICl.vol, Cl current regulated by cell volume; ClC-3, member of voltage-gated ClC Cl channel family; ICl.Ca, Cl current regulated by intracellular Ca2+ concentration ([Ca2+]i); CLCA1, member of a new Ca2+-sensitive Cl channel family (CLCA) recently cloned from human intestine (146) and mouse lung (139); ICl.ir, inward rectifying Cl current; ClC-2, member of voltage-gated ClC Cl channel family; nAE1, truncated form of anion exchange protein 1; AE3, anion exchange protein 3; ENCC1, electroneutral Na+-Cl cotransporter protein 1; ENCC3, electroneutral Na+-Cl cotransporter protein 3; M2R, muscarinic type II receptor; Gi, heterodimeric inhibitory G protein; A1R, adenosine type I receptor; AC, adenylyl cyclase; H2R, histamine type II receptor; Gs, heterodimeric stimulatory G protein; -AR, -adrenergic receptor; P2R, purinergic type 2 receptor; proposed intracellular signaling pathway for purinergic activation of CFTR (96) indicated by dashed arrows; IMAC, inner mitochondrial anion channel; VDAC, voltage-dependent anion channel. [CFTR schematic model from Welsh and Ramsey (476). Membrane topology models for ClC-2 and ClC-3 modified from Jentsch et al. (207) and include a pore-forming region between transmembrane segments 3 and 5 based on Fahlke et al. (110).]

In addition to these sarcolemmal anion channels, functional studies have provided evidence for expression of a variety of anion channels in internal membranes as well. These include a PKA-regulated anion channel in the sarcoplamic reticular membrane, two types of anion channels in the nuclear envelope, a voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, and at least two types of anion channels in the inner mitochondrial membrane that may be related to the inner mitochondrial anion conductance (IMAC) described in flux studies. A variety of sarcolemmal anion cotransporters and exchange proteins are expressed in cardiac cells, which include include Cl/HCO₃ exchange, Na⁺-dependent Cl transport, K⁺-Cl cotransport, and a novel Cl/OH exchanger.

It is becoming increasingly clear that anion channels and transport and exchange proteins in the heart mediate a variety of functions and thus play a potentially important role in cardiac physiology and pathophysiology. Because activation of sarcolemmal anion channels can significantly alter resting membrane potential and the duration of the action potential, these proteins represent novel targets for the development of new antiarrhythmic and anti-ischemic agents. Anion channels and transport proteins in the sarcolemma and internal membranes may be involved in the regulation of cell or organelle Cl activity (a_Cl), pH, volume homeostasis, and organic osmolyte transport. In many cells, there are also indications that anion transport proteins may play a role in immunological responses, cell migration, proliferation and differentiation, and possibly apoptosis (28, 239). Yet, our present understanding of the physiological significance and clinical relevance of these various anion transport pathways in the heart is incomplete. There is now well-established evidence linking several human genetic diseases to specific anion channel defects (1, 206, 249, 478), but the possible role of defects in anion channels, transporters, or exchangers in the heart to myocardial genetic diseases has not been explored.

The recent molecular identification of some of the proteins responsible for anion transport in the sarcolemma and in internal membranes of cardiac cells heralds a new era for this emerging field. Perhaps one of the greatest impediments to our present understanding of the physiological significance of anion transport proteins has been the lack of available specific pharmacological tools to investigate function. Recent studies are beginning to elucidate well-defined molecular structures for each type of anion channel and transport protein in the heart that should significantly facilitate the development of new Cl channel subtype-specific pharmacological tools for future biophysical and functional studies.

The overall aim of this review is to provide a broad overview of progress made over the past decade in the characterization of the molecular, biophysical, and pharmacological properties of anion transport proteins in heart, their species and tissue distribution, and their known or presumed physiological roles. Its content is meant to complement previously published reviews on this subject (2, 136, 162, 186, 189) and to focus on recent new developments, as well as recent controversies, in this rapidly expanding field. Although the major focus of the review is on sarcolemmal anion channels and their signaling pathways in cardiac cells, we also briefly consider the nature of anion channels in internal membranes, and electroneutral sarcolemmal anion transport and exchange proteins, and their physiological roles as well.

	II. SARCOLEMMAL CHLORIDE CHANNELS

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The first evidence for the presence of Cl channels activated by PKA (I_Cl.PKA) in cardiac cells was obtained by two groups in 1989 (13, 164, 165). The macroscopic currents recorded in guinea pig and rabbit ventricular myocytes were selective for Cl, exhibited time and voltage independence, and were blocked by anion transport inhibitors. As with Ca²⁺, K⁺, and Na⁺ channels in heart, these Cl channels were regulated by cAMP-dependent PKA phosphorylation. -Adrenergic agonists activated the channel subsequent to G protein-mediated stimulation of the cAMP pathway. Soon thereafter, the unitary currents (~13 pS) responsible for this current were identified in cell-attached membrane patches of guinea pig ventricular myocytes (101). Initially, it was not clear whether or not I_Cl.PKA in heart might have a similar molecular basis as I_Cl.PKA described in a variety of epithelial cells and known to be encoded by the CFTR gene product (186). Although the macroscopic currents in the two preparations shared a number of similar properties, the unitary currents for I_Cl.PKA in heart were linear in symmetric Cl and seemed to exhibit a much smaller conductance (102) than the larger outwardly rectifying 25- to 40-pS channels originally associated with CFTR in epithelial cells (475). However, with the successful cloning of the CFTR gene (354, 357), it soon became clear that expression of epithelial CFTR in heterologous cell systems was associated with smaller conductance (4-13 pS) channels. The demonstration that site-directed mutations of lysine residues in the transmembrane domains of CFTR resulted in dramatic changes in anion selectivity of the expressed channels provided strong evidence that CFTR functions as an anion-selective, small-conductance channel, which exhibits a linear current-voltage relationship in symmetric Cl (9, 10; see Refs. 127, 129, 353 for reviews). These data along with Northern analysis of mRNA isolated from rabbit (251) and guinea pig ventricle (304) showing hybridization using specific CFTR probes thereafter left little doubt that I_Cl.PKA in heart is due to CFTR expression.

The past 6 years have experienced an explosion of new information on the molecular, biophysical, and pharmacological properties of CFTR Cl channels and their regulation by intracellular signaling pathways. Several important reviews detailing many of these developments in cardiac (134-136, 162, 187, 189) and epithelial cells (120, 138, 379, 384, 478) have appeared. The focus of this review is to provide 1) an overview of CFTR Cl channel structure and function, regulation, species and tissue distribution, and physiological significance in heart; 2) an update of new progress made in these areas in the last few years; and 3) a consideration of some of the controversies that have emerged recently in this field in the heart.

The CFTR is composed of 1,480 amino acids, and hydropathy analysis predicts these are organized into two repeating motifs of six transmembrane spanning domains (M1-6, M7-12), two nucleotide binding domains (NBDA and NBDB), and one large regulatory (R) domain that has numerous consensus phosphorylation sites for PKA and PKC. The protein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which are structurally similar in terms of the organization of their transmembrane domains and nucleotide binding domains (170). Over 100 members of this family have been identified including P-glycoprotein (P-gp), which pumps hydrophobic compounds out of cells, and the sulfonylurea receptor (SUR), which combines with inward rectifier K⁺ (Kir6.1, Kir6.2) channel subunits to form functional K_ATP channels (5, 312). The two transmembrane motifs of ABC proteins are believed to form the pathway for solute transport, while the two nucleotide binding domains are believed to couple ATP hydrolysis to solute transport. Although CFTR seems unique in forming anion-selective channels compared with other members of the ABC superfamily, it may share some characteristic properties of ABC transporters, such as functioning as a pump for the transport of ATP as well as a regulator of other channels, such as outwardly rectifying Cl channels (ORCC) and sodium channels (78). However, whether or not CFTR transports ATP remains highly controversial (78, 346, 349, 375).

The contemporary view of CFTR channel function suggests that the highly charged R domain may represent a blocking particle, which in its unphosphorylated form keeps the channel closed, but upon phosphorylation causes channel openings via a conformational change. Phosphorylation of the R domain alone, however, is insufficient to cause channel openings, since hydrolyzable nucleotides are also required, presumably reflecting nucleotide binding to Walker A and B motifs in the NBD, which regulate channel gating properties. Thus phosphorylation of the R domain may promote ATP binding to the two NBD; however, the exact nature of the interactions between the R domain and the NBD remains unclear (78, 379). A variety of studies using site-directed mutagenesis, including scanning-cysteine-accessability analysis, have provided evidence that residues in the first (M1), fifth (M5), sixth (M6), and twelfth (M12) transmembrane spanning domains of CFTR may form part of the ion conduction pathway of the pore region (9, 49, 50, 78, 276, 288, 338, 425). The CFTR channels exhibit a lyotropic permeability sequence that favors weakly hydrated anions: SCN > NO₃ > Br > Cl > I > F (259, 490).

Although early studies suggested that the unitary and macroscopic I_Cl.PKA in heart exhibited many properties in common with epithelial CFTR channels, including similarities in rectification, anion selectivity, regulation by cAMP-dependent PKA, sensitivity to Cl channel blockers, unitary channel properties, and a dependence on hydrolyzable nucleotides for activation (13, 101, 163, 164, 191, 280, 304, 325; see Ref. 136 for review), the first molecular data on the structure of CFTR in heart came in 1993 when the cDNA encoding the 12 transmembrane spanning domains (M1-M12) were cloned and sequenced from rabbit ventricle (182). Comparison of the amino acid sequence of human epithelial CFTR with the deduced sequence from rabbit heart indicated deletion of a 30-amino acid segment in the first cytoplasmic loop of CFTR that corresponds to known locations of intron-exon junctions in human CFTR, suggesting that CFTR is an alternatively spliced (exon 5) isoform in heart. Outside of the alternatively spliced region, regions M1-M12 of the heart CFTR isoform displayed >95% identity to human epithelial CFTR. Deletion of exon 5 in the cardiac form was confirmed using Southern analysis of reverse transcription PCR products derived from canine pancreas or rabbit and guinea pig ventricle probed with oligonucleotides corresponding to nucleotide sequences specific for exon 5. The cDNA encoding the complete CFTR exon 5 isoform was subsequently cloned and sequenced from rabbit heart (158) and found to contain ~91% nucleotide sequence homology, outside of the exon 5 region, compared with human epithelial CFTR cDNA, with numerous putative PKA and PKC phosphorylation sites highly conserved in the two isoforms. Although the functional significance of exon 5 remains obscure, this region corresponds to part of the first cytoplasmic loop between M1 and M2 and does contain two putative PKC phosphorylation sites (see Fig. 4A). The cDNA encoding the rabbit cardiac exon 5 isoform was expressed in Xenopus oocytes and resulted in the appearance of I_Cl.PKA that was absent in water-injected control oocytes. This study (158) also provided evidence establishing a direct functional link between expression of CFTR and the endogenous I_Cl.PKA in native cells by showing that CFTR antisense oligonucleotides significantly reduced the density of I_Cl.PKA in acutely cultured guinea pig ventricular myocytes.

2.  Regulation

A) ADENYLYL CYCLASE/PKA. It is now well established that activation of CFTR is a two-step process requiring both PKA phosphorylation of the R domain and binding of ATP to the NBD (138, 379). In cardiac cells, numerous early studies established that I_Cl.PKA, like I_Ca and the delayed rectifier I_K (159, 287), is regulated by the adenylyl cyclase-cAMP-PKA pathway (13, 101, 163-166, 191, 264, 280, 432, 513), and the requirement for hydrolyzable nucleotides was established for I_Cl.PKA activation in heart (304) and epithelial CFTR channels (8). However, a mechanistic explanation accounting for the relationship between PKA phosphorylation of the R domain, ATP binding and hydrolysis at the NBD, and the control of CFTR channel gating properties remains elusive. This is due in part to the complicated structure of the protein, which contains at least 10 putative PKA phosphorylation sites (8 in the R domain), difficulties in demonstrating ATPase activity of the NBD biochemically, and a general lack of understanding of the dynamic interactions that may occur between the NBD and the R domain in vivo.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Schematic model of CFTR regulation by PKA phosphorylation and ATP hydrolysis. Sequential phosphorylation of 2 distinct sites or sets of sites induces a conformation change of the regulatory (R) domain, causing activation of the 2 nucleotide binding domains (NBD-A and NBD-B). The 2 phosphorylation sites are distinguished by their differential sensitivity to okadaic acid. Top row depicts closed channels states that may be dephosphorylated (D), partially phosphorylated (P1), and fully phosphorylated (P1P2). ATP hydrolysis at NBDA is associated with brief channel openings; ATP (or AMP-PNP) interactions with NBDB stabilizes the open channel state, leading to longer channel openings. [Modified from Hwang et al. (192).]

B) G PROTEINS. The role of G proteins in coupling -adrenergic receptors and muscarinic receptors to the regulation of I_Cl.PKA in heart was established in early studies. Intracellular GTP was shown to be essential for activation of I_Cl.PKA by -agonists as well as for inhibition by muscarinic agonists. The rundown of I_Cl.PKA observed in dialyzed myocytes likely reflects the loss of cellular GTP required to maintain G protein signaling mechanisms (180, 191). Indeed, cellular dialysis with GTP or use of the perforated patch technique greatly prevents rundown of I_Cl.PKA (180, 504). The effects of GTP can be attributed to convergence of G_s and G_i on adenylyl cylase, and the evidence that the same G protein-adenylyl cyclase-PKA pathway that regulates I_Ca and I_K also regulates I_Cl.PKA has been reviewed (136). There is recent data suggesting that G_s protein activation of some cAMP-independent signaling pathway, although apparently not capable of activating I_Cl.PKA in the absence of PKA phosphorylation, may play a role in amplifying the response of I_Cl.PKA to PKA (334). Because of the absence of a direct G protein effect on I_Cl.PKA, and the fact that the amplitude of I_Cl.PKA appears to reflect underlying adenylyl cyclase activity, I_Cl.PKA represents a model system for studies of receptor-G protein-adenylyl cyclase-PKA pathways in heart. I_Cl.PKA has been used to study the intracellular signaling pathways involved in the response to muscarinic (323, 324, 324, 432, 505, 507), -adrenergic (179, 196, 321), ₂-adrenergic (177), histaminergic (190, 321), purinergic (344), and endothelin (199) receptor stimulation as well as the effects of thyroid hormone (156). Regulation of I_Cl.PKA by PKC is discussed in section IIB3.

C) BASAL ACTIVITY. Unlike other cAMP-dependent channels in heart, I_Cl.PKA does not appear to be basally active in the absence of agonists, since protein kinase inhibitors generally do not appear to alter any Cl-sensitive membrane conductance (190). Whether I_Cl.PKA is basally active or not will be largely determined by the relative rates of basal adenylyl cyclase activity, basal PKA phosphorylation/dephosphorylation, as well as the level of endogenous phosphodiesterase activity in a cell. If basal PKA activity or adenylyl cyclase activity is significant, but phosphatase or phosphodiesterase activity dominates, then inhibition of endogenous phosphatases or phosphodiesterases alone should be sufficient to activate I_Cl.PKA. The initial test of this hypothesis used okadaic acid and microcystin to inhibit endogenous PP1 and PP2A in guinea pig myocytes, and these compounds failed to activate I_Cl.PKA (190). It now seems clear that this type of experiment is strongly influenced by the experimental conditions and the extent to which intracellular dialysis may dilute any resting basal adenylyl cyclase or PKA activity in the cell. Subsequent studies have shown that okadaic acid or microcystin alone (175, 306) or phosphodiesterase inhibitors like IBMX alone (163) is capable of activating I_Cl.PKA, supporting the idea that the usual absence of basal I_Cl.PKA activity may be attibutable to the predominance of basal phosphatase and/or phosphodiesterase activity in most cardiac cells. It would be interesting to test the effects of phosphatase inhibitors on I_Cl.PKA in nondialyzed cardiac myocytes using the perforated patch technique, since possible complicating effects of channel rundown may be prevented and the response to exogenously applied isoproterenol is significantly enhanced under these conditions (504).

D) TYROSINE KINASE. The role of tyrosine kinases (TK) in the regulation of epithelial CFTR Cl channels is currently under investigation, and the mechanism of activation of CFTR by the TK inhibitor genistein remains unclear. Genistein activation of epithelial CFTR Cl channels was found not to depend solely on an elevation of cAMP, suggesting some direct involvement of TK in regulation of CFTR Cl channels (194, 376). However, other explanations for the effect of genistein on CFTR channels include indirect activation of CFTR by inhibition of protein phosphatases (347, 500) and a direct, TK-independent, interaction of genistein with the CFTR Cl channel protein, possibly at a NBD (126, 467, 474). Although both cAMP-dependent and -independent mechanisms of genistein action have been described, it seems clear that the ability of genistein to modulate CFTR channels by either mechanism requires PKA prephosphorylation of CFTR; genistein has little or no effect on PKA dephosphorylated CFTR channels (126, 347, 500).

The sensitivity of I_Cl.PKA in heart to a various Cl channel antagonists is similar to epithelial CFTR channels (136, 373). Although some discrepancies have been reported, in general, I_Cl.PKA is relatively insensitive to stilbene disulfonic acid derivatives like SITS, DIDS, and DNDS but is blocked by carboxylic acid derivatives like anthracene-9-carboxylic acid (9-AC) and diphenylamine-2-carboxylic acid (DPC), arylaminobenzoates like 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), clofibric acid analogs, and sulfonylureas like glibenclamide (13, 161, 163, 386, 429, 439, 465, 499; see Fig. 3). Walsh and Wang (465) have carried out the most systematic comparison of Cl channel antagonists on I_Cl.PKA in heart and tested their specificity by simultaneously examining their effects on PKA-stimulated L-type I_Ca as well. Although both 9-AC and DPC strongly inhibited I_Cl.PKA, these compounds also blocked PKA-stimulated I_Ca, suggesting important secondary nonspecific actions of these compounds. Some of the reported variable blocking effects of 9-AC on cardiac I_Cl.PKA might also be due to an intracellular action of the compound to inhibit protein phosphatases (514). DIDS and indanyloxyacetic acid 94 (IAA-94) were poor inhibitors of I_Cl.PKA, but clofibric acid and its analogs, p-chlorophenoxy propionic acid and gemfibrozil, appeared to be the most specific inhibitors of I_Cl.PKA in guinea pig myocytes.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Structures of commonly used anion transport inhibitors. SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; DNDS: 4,4'-dinitrostilbene-2,2'-disulfonic acid; 9-AC, anthracene-9-carboxylic acid; DPC, diphenylamine-2-carboxylic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; NPBA, 5-nitro-2-(4-phenylbutylamino)benzoic acid; IAA-94, indanyloxyacetic acid.

In a recent study, the structural requirements necessary for arylaminobenzoate block of I_Cl.PKA were examined (466). Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in potency, with IC₅₀ values of 47, 17, and 4 mM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)benzoic acid, and NPPB, respectively. Further increases in carbon chain length failed to affect potency. Block by external NPPB was modulated by changes in extracellular pH, whereas block by internal NPPB was not. These results suggest that NPPB may be the most potent antagonist of I_Cl.PKA yet examined. Further structure-function studies of Cl channnel antagonists on I_Cl.PKA offer potential for the discovery of new potent antagonists that might exhibit a higher degree of selectivity among the different types of Cl channels present in cardiac muscle.

Electrophysiological studies indicate a significant species and tissue variability in the expression of I_Cl.PKA. In general, I_Cl.PKA is most often found in adult ventricular, but not in atrial or sinoatrial nodal cells in guinea pig, rabbit, and cat (164, 427, 451, 513). In contrast, no evidence for I_Cl.PKA has yet been found in adult canine (404), rat (98, 212), or mouse hearts (252); however see sect. IIE), although I_Cl.PKA has been reported in rat (436) and mouse (40) neonatal myocytes, suggesting that in some species I_Cl.PKA may be developmentally regulated. Evidence for functional expression of I_Cl.PKA in human heart is controversial (see sect. IIA5B). Density of I_Cl.PKA is higher in epicardial compared with endocardial cells in rabbit ventricle (427), and a recent study using in situ hybridization with CFTR specific probes combined with electrophysiological measurements of I_Cl.PKA density has confirmed this pattern of expression in rabbit ventricle (444).

Because early studies generally failed to find I_Cl.PKA in atrial myocytes, this has led to the notion that I_Cl.PKA may have physiological relevance only in the ventricle. However, a small percentage of guinea pig atrial myocytes has been reported to express I_Cl.PKA (282). In a timely study, James et al. (198) quantitated mRNA levels of CFTR in guinea pig atrium and ventricle and found strong correlations with I_Cl.PKA densities, measured electrophysiologically. Specifically, mRNA levels and I_Cl.PKA densities were lower (but not absent) in atrial cells and highest in ventricular epicardial cells compared with endocardial cells. This study set a new standard for quantitative mRNA studies in heart, and similar studies combining membrane current densities with quantitative RT-PCR of CFTR gene products in other species are needed to determine the generality of this pattern of tissue-specific myocardial expression of CFTR.

In earlier studies, RT-PCR using primers designed to amplify several different regions of CFTR was used to characterize CFTR expression in different species and areas of the heart (182, 251, 471). These results are illustrated in Figure 4. Of the three different regions of CFTR that were amplified, those corresponding to NBDA (E9-E13', 550 bp) and M7-M12 (E14-E17', 944 bp) were detected in ventricular tissue of rabbit and guinea pig heart and in atrium and ventricle of both human and simian hearts. Amplification of these products from dog atrium and ventricle and guinea pig and rabbit atium was not detected. These RT-PCR reactions were carried out in a single 30-cycle amplification, in contrast to James et al. (198) in which two amplifications generating extremely high sensitivity were performed. The lack of detectable CFTR expression in canine heart is consistent with the results of electrophysiological studies that have failed to observe I_Cl.PKA in similar preparations (88, 404). Surprisingly, in virtually every cardiac tissue in which PCR was performed, regions corresponding to M1-M6 (E3-E7') could be amplified to detectable levels. In all animal species, only a 681-bp product was detected, indicating exclusive expression of the exon 5 isoform, compared with control dog pancreas tissue in which the epithelial exon 5+ transcript (771 bp) is known to be expressed. Interestingly, in human atrium and ventricle and simian ventricle, both exon 5 and exon 5+ transcripts appear to be expressed. The detection of CFTR amplification products corresponding to M1-M6 segments of CFTR in tissues in which I_Cl.PKA is not detected (e.g., canine) prompted speculation that since this region of CFTR is believed to contribute to the channel pore (see sect. IIA1), such anomolous expression may be due to sequence homology of a conserved pore region in other types of Cl channels in heart (187). Although this remains a possible explanation, especially given the variety of different types of Cl channels that appear to be expressed in intracellular membranes of cardiac cells (see sect. VI), considerable future effort is needed to reconcile these apparently inconsistent expression patterns of CFTR thus far revealed by electrophysiological and molecular studies. It is possible that pseudogenes give rise to variant truncated transcripts for CFTR. Reverse transcription-polymerase chain reaction experiments designed to amplify CFTR specific segments that extend further than exon 7 were unsuccessful (Horowitz, unpublished observations). Future studies should include 1) a more extensive examination of whether or not I_Cl.PKA can be detected in canine myocardial tissue and in atrial tissue of several species, 2) the use of quantitative RT-PCR to clearly establish relative CFTR mRNA levels, and 3) the use of in situ hybridization and/or immunocytochemical techniques to clearly distinguish sarcolemmal CFTR expression from expression in internal membranes.

View larger version (54K): [in this window] [in a new window]   Fig. 4. RT-PCR amplification of heart-derived cDNA encoding NH2-terminal (681 bp), nucleotide binding domain A (NBDA, 550 bp), and COOH-terminal (944 bp) segments of CFTR. A: predicted membrane topology of CFTR indicating the transmembrane segments, nucleotide binding domains (NBDA and NBDB), and regulatory domain (R). Oligonucleotide primers were designed to hybridize to sequences in exon 3 (sense) nucleotides (nt) 270-295 and exon 7 (antisense) nt 926-951 of CFTR cardiac (accession no. U40227) for the NH2-terminal region (E3-E7'), exon 9 (sense) nt 1379-1399 and exon 13 (antisense) nt 1900-1929, for the NBDA region (E9-E13'), and exon 14 (sense) nt 2661-2681, exon 17 (antisense) nt 3585-3605 for COOH-terminal region (E14-E17'). B: representative agarose gel with amplification products from the RT-PCR reactions. Data demonstrate that rabbit, guinea pig, and canine hearts result in amplification of only the exon 5-deleted product (681 bp), whereas human and simian heart tissues yield both the exon 5-deleted and nondeleted (771-bp) products. RT-PCR amplification of mRNA from canine pancreas tissue only yielded the nondeleted product. Amplification products for other regions of CFTR are only detectable in rabbit and guinea pig ventricle, human atrium and ventricle, and simian ventricle. [Data compiled from Horowitz and co-workers (182, 187, 471).]

5.  Recent controversies

A) Na⁺DEPENDENCE In the original description of an isoproterenol-induced Na⁺-dependent current, Na⁺ was concluded to be a major charge carrier of the current since removal of extracellular Na⁺ attenuated the response (99, 100). This Na⁺ sensitivity was subsequently verified in other studies (163, 280), but rather than indicating substantial Na⁺ permeability of the channels, it appeared to involve alteration of the I_Cl response at a regulatory site in the cAMP-dependent pathway. Attenuation of I_Cl by reduction of extracellular Na⁺ was not accompanied by any significant change in the current reversal potential (163, 280), and a similar sensitivity to extracellular Na⁺ was shown for -adrenergic regulation of I_Ca (281). A later examination of the extracellular Na⁺ sensitivity of I_Cl.PKA suggested that it may be modulation by Na⁺ at an intracellular site, possibly involving phosphorylation or dephosphorylation of Cl and Ca²⁺ channels (167). However, later key studies helped to eventually resolve the issue. Tareen et al. (433) suggested that most of the apparent extracellular Na⁺ sensitivity occurs due to antagonism between Na⁺ substitutes and isoproterenol at the level of the -adrenoreceptor, since they could not observe Na⁺ modulation using agents that activate the pathway beyond the -receptor. Studies by Zakharov et al. (506) also showed that the observed extracellular Na⁺ sensitivity may be related to muscarinic agonist activity of the Na⁺ substitutes (Tris or tetramethylammonium) used earlier, thus leading to inhibition of adenylate cyclase activity via G_i protein activation. A recent study has confirmed that once these effects are prevented, changes in extracellular or intracellular Na⁺ have no direct effect on I_Cl.PKA (472).

B) FUNCTIONAL EXPRESSION IN HUMAN HEART. The molecular evidence presently available strongly suggests that CFTR message is expressed in both atrial and ventricular human myocardium (251, 471). In fact, RT-PCR products representing four distinct regions of CFTR all suggest expression of CFTR in both human as well as simian atrium and ventricle (Fig. 4). Moreover, in contrast to all other animal species yet examined, there is evidence for expression of both the exon 5+ as well as the exon 5 isoforms in human and simian myocardium. However, electrophysiological evidence for functional expression of CFTR Cl channels in human heart is weak. Only one study has provided evidence for the existence of I_Cl.PKA in human myocytes (471), and that evidence was limited by the fact that only 27% of the atrial myocytes examined (average patient age 62 years) exhibited an intact adenylyl cyclase/PKA pathway (as assessed by measuring the response of I_Ca to forskolin). Of these, 63% responded to forskolin with the activation of a time-independent I_Cl that was DIDS insensitive. Consistent activation of I_Cl.PKA by forskolin was observed in every simian ventricular myocyte examined. In 3 of 12 giant excised human atrial patches examined, unitary Cl channels activated by PKA catalytic subunit with a mean slope conductance of ~14 pS were observed. DIDS insensitivity, a 8- to 14-pS single-channel conductance, activation by PKA, and a linear current-voltage relationship in symmetrical Cl are all properties characteristic of cardiac and epithelial CFTR Cl channels (136, 353, 478), and inconsistent with the known properties of most other types of Cl channels in heart, including I_Cl.vol (see Table 1).

View this table: [in this window] [in a new window]   Table 1. Properties of functionally identified sarcolemmal Cl channels in heart

C) FUNCTIONAL SIGNIFICANCE OF EXON 5. Existing molecular evidence suggesting exclusive expression of the exon 5 isoform of CFTR in the heart of most animal species examined to date raises the obvious question of functional significance. Four cytoplasmic loops (CL) (ignoring the large NBDA and R-domain region) connect the transmembrane domains of CFTR (Fig. 4), which are expected to be ~55-65 amino acids in length and generally are highly conserved between different species (79, 354). It has been suggested that due to their highly lipophilic nature, the CL may interact with other regions of CFTR or other proteins (430), but the functional significance of the CL is only beginning to be understood. Exon 5 encodes 30 amino acids in first cytoplasmic loop (CL1), but their functional role is unknown. On the basis of mutagenesis experiments, CL2 and CL3 have been proposed to help stabilize the full conductance state of CFTR (378, 492), whereas CL4 appears to affect the responsiveness to regulatory stimuli (377). It has been reported that an engineered epithelial exon 5 isoform of CFTR fails to generate functional channels when expressed in HeLa cells, presumably due to defective intracellular processing, suggesting that exon 5 transcripts may generate nonfunctional proteins (77). In addition, exon 5 isoforms were found to be the most abundant alternatively spliced transcripts in mice. A subsequent study confirmed that the engineered epithelial exon 5 isoform exhibited a processing defect, becoming trapped in intracellular membranes in HEK 293 cells, but retained some functional Cl channel activity when isolated and incorporated into lipid bilayer membranes (493). These exon 5 CFTR channels exhibited an average P_o significantly smaller (P_o < 0.01) than wild-type channels (P_o ~0.3), and channels exhibited a small subconductance state (2-3 pS) more frequently compared with wild-type channels. These results suggest that CL1 may be involved in both intracellular processing as well as the conductance properties of the channel.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Recombinant rabbit cardiac exon 5 CFTR channels in inside-out (AC) and cell-attached (D and E) membane patches from Xenopus oocytes. In inside-out patches, channels were initially activated by 100 nM PKA catalytic subunit (100 nM) and 0.5 mM MgATP (A). Channels closed upon washout of PKA and MgATP but could be reopened by exposure to MgATP alone. Representative channel openings at different patch potentials (Vp) in the presence of PKA and MgATP are shown in B, and single-channel conductance in this patch was 7.2 pS (C). In cell-attached membrane patches, CFTR channels were opened by bath application of 1 mM forskolin (FSK; D) or 100 nM phorbol 12,13-dibutyrate (PDBu) (E). Mean single-channel conductance for forskolin-activated channels was 9.5 ± 0.8 pS (n = 5) and for PDBu-activated channels was 10.6 ± 0.4 pS (n = 5) (F). For cell-attached patches, pipette solution contained (in mM) 100 N-methylglucamine chloride (NMG-Cl), 5 CsCl, 2.5 MgCl2, and 10 HEPES, pH 7.3; bath solution contained modified ND-96 solution (in mM: 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 100 niflumic acid, and 5.0 HEPES, pH 7.4). For inside-out patches, pipette solution (external solution) had same composition as bath solution for cell-attached mode; bath (internal) solution contained (in mM) 100 NMG-Cl, 6 CsCl, 2 MgCl2, 5 EGTA, and 10 HEPES, pH 7.3.