Perspective Nucleotides and Ion Channel Regulation P. E. Nasmith and Dale J. Benos Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama

Signal transduction by hormones, neurotransmitters, and growth factors is almost invariably associated with changes in the activity of one or more ion channels in the plasma membrane of cells. Alterations in receptors and channels are implicated in the pathophysiology of many disease states. At least three hereditary diseases (cystic fibrosis, hyperkalemic periodic paralysis, and malignant hyperthermia) have been linked to defects in channels for Cl, Na-, and Ca2+, respectively (1-3). Therefore, knowledge of channel structure and regulation is required for understanding ion channel dysfunction in disease as well as in normal states. Further, this knowledge is also essential for devising strategies for pharmacologic and/or immunologic intervention to help curb the extent of disease processes. The mechanisms by which extracellular signals regulate the opening and closing of various ion channels are diverse. Three broad classes of ion channels have been described: (1) voltage-gated channels, regulated by changes in transmembrane potential; (2) ligand-gated channels, regulated by binding of an extracellular ligand to the channel; and (3) second messenger-gated channels, regulated by changes in intracellular ions or co-factors stemming from activation of cell surface receptors. A wide variety of regulatory mechanisms has been described for channels falling into the last group, including stimulation by G proteins, phosphorylation by protein kinases, and binding by arachidonic acid, inositol phosphates, intracellular Ca2+, and nucleotides (4-7). To understand the molecular mechanisms underlying selective ion transport by channels, it is essential to elucidate the primary structure of the constituent protein(s) and to understand the control pathways involved in regulating channel function. Several approaches have been used to study the structure and functional regulation of ion channels. Electrophysiologic methods and the use of isotopic ion flux measurements have the advantage that functional studies can be carried out in vivo, without removing the channel from its native environment. However, it is impossible to identify from these studies alone the protein(s) involved in the observed activity. Biochemical methods directed at purification (Received for publication March 6, 1992) Address correspondence to: Dale 1. Benos, Ph.D., Department of Physiology and Biophysics, University of Alabama at Birmingham, DAB Station, Birmingham, AL 35294. Abbreviations: cystic fibrosis transmembrane conductance regulator, CFTR; Madin-Darby canine kidney cells, MOCK cells. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 567-568, 1992

of the channel protein itself are difficult for several reasons. With some exceptions, channel proteins form a low percentage of total cellular protein, limiting the amount of material available. Because all channels are membrane proteins, it is difficult to solubilize them without denaturation and at least some loss of activity. If no specific high-affinity ligands exist for the channel of interest, activity must be assayed by incorporation into vesicles or artificial bilayers, a process that can consume large amounts of the protein. More recently, molecular biologic techniques have been successfully combined with electrophysiologic and biochemical techniques to elucidate the structure/function relations of ion channels. Once a channel has been cloned, it can be expressed in a cell type free of similar channels, such as the Xenopus oocyte expression system (8, 9). Expression of the cloned protein allows the identification of which subunits are sufficient for ion transport. The amino acid sequence of a channel may itself suggest possible modes of regulation, through the presence of consensus sequences for phosphorylation or nucleotide binding. Site-directed mutagenesis and deletion studies can identify which regions of a channel protein are important in pore formation and regulation of channel opening and closing. Knowledge of the amino acid sequence and the use of expression systems to produce large amounts of the protein greatly facilitates the production of antibodies. Immunoprecipitation of channels from their native cells provides a means to identify associated (and potentially regulatory) proteins, as well as a means for detecting biochemical changes in the channel itself (such as phosphorylation) resulting from cellular stimulation. Historically, the majority of work has focused on nerve and muscle cells, but more recently these techniques are being used to examine ion channels in other tissues, including epithelia. Expression of cloned channels has been instrumental in dissecting the regulation of channels by second messengers, because a single channel can be expressed as the predominant current within a cell and second messengers selectively stimulated or added intracellularly. One important area of study is the requirement for and role of nucleotides in channel activity, both directly and indirectly, by increasingly diverse mechanisms. Indirectly, cytoplasmic ATP and GTP modulate ion channel activity through their roles as substrates for protein kinases and G proteins, respectively. Phosphorylation by protein kinases has been reported to modulate Na-, K+, Cl-, and Ca2+ currents (6). Activation of G proteins by cell surface receptors requires binding of GTP by the a subunit, and a-GTP complexes can directly activate



opening of acetylcholine-sensitive atrial K+ channels, as well as other channels (4). Several types of ion channels are regulated by binding of nucleotides directly to the channel. An increase in intracellular ATP results in the closing of K+ channels in heart, brain, and pancreatic {3 cells (7). ATP is thought to act directly on the channel, with no requirement for kinase activity. ATP-gated closing of the channel in {3 cells and the consequent depolarization of the membrane is thought to be the mechanism by which glucose, through its intracellular metabolism to ATP, controls insulin secretion. Cation channels cloned from photoreceptors and olfactory epithelium are opened by binding of cGMP or cAMP to cytoplasmic sites in the channel protein (8, 10). Stimulation of photoreceptor cells by light results in hydrolysis of cGMP and closing of the channel, while in olfactory epithelium activation of odorant receptors is thought to result in an increase in cAMP and opening of a cation channel (8, 10). The role of nucleotides in the regulation of CI- transport by the cystic fibrosis transmembrane conductance regulator (CFTR) is complex, with both direct and indirect requirements for ATP (1). CFTR contains two predicted cytoplasmic nucleotide binding domains, as well as a third domain containing consensus sites for phosphorylation. CI- transport by CFTR requires both phosphorylation by protein kinase A and the presence of hydrolyzable nucleotides, which presumably interact at the nucleotide binding domains (1). Thus, even for a single channel, multiple requirements for nucleotides may exist. Recently, a novel voltage-activated CI- channel has been cloned from Madin-Darby canine kidney (MDCK) cells, an epithelial cell line derived from dog kidney (9). Inspection of the amino acid sequence suggested that a consensus site for nucleotide binding lay near the outside mouth of the channel. When the channel was expressed in Xenopus 00cytes, whole cell currents were decreased in the presence of a variety of extracellular nucleotides, of which cGMP and cAMP were the most potent. Extracellular nucleotides have previously been demonstrated to influence channel opening through their interaction with cell surface purinergic receptors (5), but the closing of channels through direct binding to extracellular sites on the channel has not been investigated. Winter and associates report in this issue of the journal (11) that the tetradecapeptide mastoparan stimulates K+and CI- currents in the apical membrane of MDCK kidney epithelial cells. Mastoparan is reported to interact with and stimulate the ex subunit of some G proteins (12, 13). Mastoparan activation did not correlate with increased cAMP, intracellular Cav, diglyceride, or arachidonic acid in the MDCK cells, indicating that activation of CI- and K+

currents was not mediated by any of these second messengers. In MDCK monolayers where the basolateral membrane was permeabilized to deplete cells of endogenous GTP, the effect of mastoparan was dependent on the addition ofGTP')'S. The investigators suggest that mastoparan may be acting through a G protein to cause direct activation of K+ and CI- channels in the apical membrane. The distribution of ion channels in the apical versus basolateral membrane of MDCK and other epithelial cells is known to be markedly different (14). This asymmetric distribution is essential for secretory and/or absorptive function. Selective permeabilization of one membrane allows manipulation of cytoplasmic parameters, for example nucleotide concentrations, while retaining intracellular proteins. When combined with studies on isolated epithelial cells (single-channel recordings in excised patches of membrane, expression studies of cloned channels), a clearer understanding of epithelial function in normal and diseased tissue will be obtained. References 1. Anderson, M. P., H. A. Berger, D. P. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67:775-784. 2. Rojas, C. V., J. Wang, L. S. Schwartz, E. P. Hoffman, B. R. Powell, and R. H. Brown, Jr. 1991. A met-to-val mutation in the skeletal muscle Na" channel a-subunit in hyperkalaemic periodic paralysis. Nature 354:387-389. 3. Gillard, E. F., K. Otsu, J. Fujii et al. 1991. A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia. Genomics 11:751-755. 4. Brown, A. M., and L. Birnbaumer. 1990. Ionic channels and their regulation by G protein subunits. Annu. Rev. Physiol. 52: 197-213. 5. Linden, J. 1991. Structure and function of Al adenosine receptors. FASEB J. 5:2668-2676. 6. Levitan, I. B. 1988. Modulation of ion channels in neurons and other cells. Annu. Rev. Neurosci. 11:119-136. 7. Ashcroft, F. M. 1988. Adenosine 5'-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11:97-118. 8. Kaupp, U. B., T. Niidome, T. Tanabe et al. 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342:762-766. 9. Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. A novel mammalian chloride channel identified by expression cloning. Nature 356:238-241. 10. Dhallan, R. S., Y. King-Wai, K. A. Schrader, and R. R. Reed. 1990. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347:184-187. 11. Winter, M. c., M. R. Carson, R. A. Sheldon, and D. M. Shasby. 1992. Mastoparan activates apical chloride and potassium conductances, decreases cell volume, and increases permeability of cultured epithelial cell monolayers. Am. J. Respir. Cell Mol. Bioi. 6:583-593. 12. Higashijima, T., 1. Bumier, and E. M. Ross. 1990. Regulation of G, and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. J. Bioi. Chem. 265:14176-14186. 13. Weingarten, R., L. Ransnas, H. Mueller, L. A. Sklar, and G. M. Bokoch. 1990. Mastoparan interacts with the carboxy terminus of the a subunit of G j • J. Bioi. Chem. 265:11044-11049. 14. Fuller, C. M., and D. 1. Benos. 1991. The physiology and biochemistry of sodium and chloride permeability pathways in epithelia. J. Nutr. Biochem. 2:348-362.

Nucleotides and ion channel regulation.

Perspective Nucleotides and Ion Channel Regulation P. E. Nasmith and Dale J. Benos Department of Physiology and Biophysics, University of Alabama at B...
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