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Cultured mouse MTAL cells contain more mRNA encoding the Cl- channel mcCIC-Ka, which mediates CTAL Cl- absorption, than mRNA encoding the Cl- channel mmCIC-Ka, which mediates MTAL Cl- absorption. mmCIC-Ka and mcCIC-Ka have three functional differences: 1) mmCIC-Ka open time probability, Po, increases with increasing cytosolic Cl-, but variations in cytosolic Cl- do not affect Po in mcCIC-Ka; 2) mmCIC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on Po in mcCIC-Ka; and 3) mmCIC-Ka channels have single-ion occupancy, while mcCIC-Ka channels have multi-ion occupancy. Using basolateral vesicles from MTAL cells fused into bilayers, we evaluated the effects of 1 mM cytosolic phenylglyoxal (PGO), which binds covalently to lysine or arginine, on Cl- channels. With PGO pretreatment, Cl- channels were uniformly not gated either with increases in cytosolic-face Cl- or with (ATP + PKA) at 2 mM cytosolic-face Cl-; and they exhibited multi-ion occupancy kinetics typical for mcCIC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl- access to arginine or lysine residues on mmCIC-Ka by PGO results in Cl- channels having the functional characteristics of mcCIC-Ka channels.

We evaluated the effects of culturing mouse MTAL cells under conditions that suppressed steady-state cytosolic Cl- on chloride channels fused into bilayers from basolateral vesicles of cultured MTAL cells. We used two agents to suppress Cl- entry: 10(-6) M PGE2 and 10(-4) M bumetanide. Basolateral Cl- channels from control cultured MTAL cells exhibited the signature characteristics of mmCIC-Ka channels: increased open-time probability (Po) either by raising cytosolic-face [Cl-] or, at 2 mM cytosolic Cl-, by adding (ATP + PKA), and first-order conductance kinetics. Either 10(-6) M PGE2 or 10(-4) M bumetanide in culture media reduced steady-state MTAL cytosolic Cl-. Chloride channels from these cells exhibited characteristics unique to CTAL mcCIC-Ka channels, namely: no augmentation of Po either by raising cytosolic Cl- or with cytosolic (ATP + PKA), and multi-ion occupancy. Semi-quantitative RT-PCR and real-time quantitative PCR showed that culturing MTAL cells with 10(-6) M PGE2 or 10(-4) M bumetanide reduced mRNA levels encoding mmCIC-Ka but not mRNA levels encoding mcCIC-Ka. However, when MTAL cells were cultured under control conditions, and then pre-incubated for 60 minutes with 10(-4) M bumetanide, cytosolic Cl- fell acutely but Cl- channels exhibited characteristics of mmCIC-Ka channels. Thus PGE2 and bumetanide, both of which lower steady-state MTAL cytosolic Cl- concentrations, inhibit either the transcriptional and/or the translational processes for mmCIC-Ka synthesis.

This paper describes the kinetics of Cl- flux through mcClC-Ka Cl- channels from basolateral membranes of mouse CTAL cells. We have cloned two separate but highly homologous Cl- channels, mmClC-Ka from cultured mouse MTAL cells and mcClC-Ka from cultured mouse CTAL cells. The mmClC-Ka and mcClC-Ka channels appear to mediate net Cl- absorption in the MTAL and CTAL, respectively. The kinetics of Cl- permeation through mmClC-Ka channels exhibit traditional criteria for a first-order process, including saturation kinetics. Thus mmClC-Ka channels operate functionally as if the channels were occupied by a single Cl- ion at any given time. In the present studies, we examined conductance-concentration relations in mcClC-Ka channels, and compared both mole-fraction effects and ion selectivity characteristics in mmClC-Ka and mcClC-Ka channels. In mcClC-Ka channels, we observed both self-block at high external Cl- concentrations and, at constant ionic strength, an anomalous mole-fraction effect using external solutions containing varying F-/Cl- concentrations. Neither effect was obtained in mmClC-Ka channels. These data are consistent with the possibility that Cl- permeation through mcClC-Ka channels involved multi-ion occupancy channels that expressed single-file behavior.

Background: Our prior data indicate that two separate but homologous basolateral chloride (Cl-) channels, mmClC-Ka and mcClC-Ka, are the principal mediators of net Cl- absorption in mouse medullary thick ascending limb (MTAL) and cortical thick ascending limb (CTAL) cells, respectively. In the present studies, we evaluated the possibility that there might be translational or post-translational suppression of mmClC-Ka and mcClC-Ka activity in CTAL and MTAL cells, respectively., Methods: Polymerase chain reaction (PCR) fragments were prepared that were highly specific for either mmClC-Ka or mcClC-Ka, the cDNAs encoding mmClC-Ka and mcClC-Ka, respectively., Results: Using reverse transcription (RT)-PCR with these highly specific products, mRNAs specific for non-homologous channel sequences in either mmClC-Ka or mcClC-Ka were present in both MTAL and CTAL cells., Conclusions: Both mouse MTAL and CTAL cells contain the mRNAs encoding mmClC-Ka and mcClC-Ka. There may be translational or post-translational suppression of mmClC-Ka activity in CTAL cells, and of mcClC-Ka activity in MTAL cells.

Cl- transport in the loop of Henle is responsible for reclamation of 25-40% of the filtered NaCl load and for the formation of dilute urine. Our understanding of the physiologic and molecular mechanisms responsible for Cl- reabsorption in both the thin ascending limb and thick ascending limb of Henle's loop has increased greatly over the last decade. Plasma membrane Cl- channels are known to play an integral role in transcellular Cl- transport in both the thin and thick ascending limbs. This review focuses on the functional characteristics and molecular identities of these Cl- channels, as well as the role of these channels in the pathophysiology of disease.

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl(-) channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites-threonine 185, threonine 187 and serine 270-which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P(o) in basolateral CTAL Cl(-) channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T(1/2), sec) for (36)Cl(-) efflux in cultured MTAL cells from 82.4 +/- 6.8 sec (sem) to 187.8 +/- 9.5 sec (n = 5; P = 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T(1/2) for (36)Cl(-) efflux from 80.9 +/- 6.3 sec to 191.8 +/- 6.5 sec (n = 5; P = 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl(-) channels mediating net Cl(-) absorption in mouse MTAL and CTAL, respectively.

The purpose of this brief commentary is to provide a description of how renal physiology, and more particularly, renal tubular physiology, has evolved over the past thirty years, since the occasion, obviously, is a celebration of the Thirtieth Course on Advances in Nephrology and Dialysis. My arguments will begin by quoting from Homer Smith's book, The Kidney, and merging Smith's observations, which were integrative in the sense that they did not specify detail, with detailed incursions into tubular physiology, first at a cellular level and then at a molecular level. For convenience, the nephron is divided into four functional segments: the proximal nephron; the ascending limb; the distal nephron; and the collecting duct. Each of these carries out a specific function. The proximal nephron absorbs about two-thirds of filtered sodium, without dissociating salt and water absorption. The thick ascending limb absorbs 25% of filtered Na+, but no water. The distal nephron absorbs 10% of filtered Na+ in close relation with K+ and, to some extent, H+ secretion. Finally, the collecting duct includes three kinds of cells: the cortical collecting duct, which is responsible not only for Na+ absorption and K+ secretion, but also for the bulk of the absorption of free water; the outer medullary collecting duct (OMCD), which is largely responsible for the final steep drop in urine pH which occurs between cortex and papilla; and the inner medullary collecting duct (IMCD), whose major function is the final absorption of approximately 5% of filtered Na+.

Background: This article reports studies on the kinetics of chloride (Cl-) conductance in Cl- channels fused into bilayers from basolaterally enriched vesicles from rabbit outer medulla. A considerable body of evidence indicates that these channels represent rbClC-Ka, a 77 kDa kidney-specific protein of the ClC family of Cl- channels. rbClC-Ka, a candidate channel for mediating net Cl- absorption in the medullary thick ascending limb (MTAL), has been cloned from rabbit outer medulla and localized by immunofluorescence to basolateral membranes of the MTAL. Thus, this is the first account, to our knowledge, of the kinetics of ion permeation through a renal Cl- channel mediating net basolateral Cl- absorption in the thick ascending limb of Henle (TALH), and this channel may represent rbClC-Ka., Methods: The electrophysiological properties of these channels were studied by fusing basolaterally enriched MTAL vesicles into planar bilayer membranes., Results: Cl- conductance through these channels was concentration dependent and saturable. The relationship between gCl (pS) and symmetrical aqueous Cl- concentrations could be expressed in terms of the Michaelis equation with a limiting conductance (GClmax, pS) of 114 pS at infinitely high aqueous Cl- concentrations and a K1/2 of 163 mM Cl-. A log-log plot of the conductance-Cl- concentration relations, in the nonsaturating Cl- concentration range, had a slope of 0.91, that is, virtually unity. The relatively impermeant anion I- produced a voltage-dependent conductance blockade that could be overcome at high electric field strengths., Conclusions: The experimental data described earlier here fulfill the traditional criteria for a first-order process with a single Cl- ion occupying these channels at a given time. Although the channels may contain multiple ion binding sites, the latter function, in integral kinetic terms, as a single rate-limiting locus.

Background: Antidiuretic hormone (ADH) or adenosine 3', 5'-cyclic phosphate (cAMP) analogues augment net NaCl absorption in microperfused mouse medullary thick ascending limb (MTAL) segments but not in cortical thick ascending limb (CTAL) segments. This ADH-dependent MTAL effect is due to increased apical Na+/K+/2Cl- admittance and apical K+ recycling accompanied by a rise in calculated intracellular Cl- concentrations and by a threefold rise in basolateral Cl- conductance. rbClC-Ka, a 75.2 member of the ClC family of Cl- channels, mediates net Cl- absorption in the MTAL. The gating characteristics of rbClC-Ka channels from their intracellular surfaces are, to our knowledge, unique among Cl- channels. The channels are activated by small increases in intracellular Cl- (K1/2 = 10 mM Cl-). Adenosine triphosphate plus the catalytic subunit of protein kinase A (ATP + PKA) gate rbClC-Ka when cytosolic Cl- concentrations are 25 mM. Thus, in mouse MTAL segments, ADH-dependent rises in cytosolic Cl- are primarily responsible for basolateral Cl- conductance increases., Methods: These experiments compared the properties of Cl- channels fused into bilayers from basolaterally enriched vesicles from cultured mouse CTAL cells with rbClC-Ka channels., Results: The key findings were that anti-rbClC-Ka, antibody that recognizes and blocks rbClC-Ka, recognized and blocked basolateral Cl- channels in CTAL cells, that the extracellular faces of the CTAL channels were, like rbClC-Ka, substrate gated with a K1/2 of approximately 170 mM Cl-, and that, unlike rbClC-Ka channels, cytosolic faces of basolateral CTAL Cl- channels were not gated by either increasing cytosolic Cl- concentrations or cytosolic (ATP + PKA). This failure of activation of basolateral CTAL Cl- channels was confirmed using excised patch clamp studies. Finally, on Western blots, anti-rbClC-Ka recognized a 74 kDa band on basolateral CTAL vesicles., Conclusions: Basolateral CTAL Cl- channels probably share a high degree of structural homology and possibly molecular mass with rbClC-Ka channels. However, significant differences between rbClC-Ka channels and CTAL Cl- channels account for the inability of increasing either cytosolic Cl- or (PKA + ATP) to raise Po in CTAL basolateral Cl- channels.

This article summarizes briefly some factors responsible for edema in chronic congestive heart failure. It is now generally thought that so-called 'backward failure' is a manifestation of diastolic dysfunction, while systolic 'pump failure' is a disease that depends on two key factors: an inadequate cardiac output, and renal salt and water retention. The key elements involved in what might be termed the 'integrated volume response' are hemodynamic and renal factors. The hemodynamic factors include vasoconstriction, tachycardia and a reduced venous capacitance. These responses occur within minutes, while salt and water retention occurs over days to weeks. The key renal elements modulating sodium retention in congestive heart failure include, at a minimum, four variables. First, there is a reduction in renal blood flow produced by the almost simultaneous operation of alpha- and beta-catecholamines, antidiuretic hormone, the endothelins, and angiotensin II. Second, activation of the tubuloglomerular feedback system enhances intrarenal angiotensin II release, which augments proximal sodium absorption. In addition, beta-catechols also enhance proximal sodium absorption. A third key element involved in renal sodium retention is activation of apical sodium channels, ENaC, of principal cells in the cortical collecting tubule by aldosterone and by vasopressin. Finally, the inner medullary collecting duct becomes resistant to the action of atrial natriuretic peptide, thus adding a final dimension to the syndrome of sodium retention in underfilling.

Extracellular fluid volume is determined by sodium and its accompanying anions. There are control mechanisms which regulate sodium balance in the body. These include high and low pressure baroreceptors, intrarenal baroreceptors, renal autoregulation, tubuloglomerular feedback, aldosterone, and numerous other physical and hormonal factors. Sodium transport by the nephron involves active and passive processes which occur in several different nephron segments. Mechanisms of cotransport, Na(+)-H+ exchange, antiporters and ion-specific channels are all utilized by the nephron to maintain sodium balance. These regulatory factors and transport mechanisms for sodium in the kidney will he discussed in detail.

Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.

Chloride transport by the medullary thick ascending limb is a major determinant of urinary concentrating and diluting power and also of tubuloglomerular feedback. In this review, we discuss the electrophysiologic, regulatory, and molecular features of a Cl- channel present in basolateral membranes from the medullary thick ascending limb. Functional and immunohistochemical data support the view that rbCIC-Ka, a CIC Cl- channel cloned from rabbit kidney medulla, may be the Cl- channel which mediates basolateral Cl- efflux.

What I have tried to describe in these brief introductory statements are the key elements stimulating renal sodium retention mediated by arterial underfilling, as illustrated by the volume repletion reaction (Fig. 3). They may be summarized by saying that edematous states characterized by underfilling-most notably systolic pump failure-represent, in fact, suicidal arterial filling. This point of view is illustrated in Figure 8. Essentially, as shown in Figure 8, the original reduction in filling of the arterial tree, by provoking both hemodynamic changes and diminished sodium avidity, has the net effect of increasing end diastolic volume and, for a time, cardiac output, but at two great expenses: an increased afterload and a an increased preload. In the end, these latter two effects produce a reduced ejection fraction which obviously propagates the syndrome of systolic failure. Thus, suicidal arterial filling represents a vicious cycle in which homeostatic mechanisms set into play an attempt to compensate for inadequate arterial filling, which inevitably leads to increasing degrees of cardiac dysfunction and, hence, to deterioration of the patient. I have enjoyed presenting these introductory remarks. I am certain that subsequent talks in this Conference will consider more explicitly the provocative issue of the "overfilling" hypothesis as a mechanism for sodium retention in edematous states such as the nephrotic syndrome and cirrhosis.

It is reasonable to argue that Ike's contributions mirror closely the extraordinary growth and development of the ISN in the last two decades. As we noted at the outset of this article, Ike's tenure on the Executive Committee is the longest tenure of any individual to date. Further, it is improbable, given the current structure of the Executive Committee, that the duration of his tenure will be equaled, at least in the near future. More importantly, one can measure the impact of his tenure in terms of formidable accomplishments. These include, at a minimum, the development of Kidney International both as an academic triumph and as a fiscally sound organ; the initial formulation of a reasonable and apparently enduring model for the Forefronts in Nephrology series; and the transformation of the ISN Congresses to their current format. Lastly, we should stress another contribution of Ike's to the ISN, clearly palpable to those of us who have served on the Executive Committee. That is, his consistent wisdom and diplomacy, his dedicated preoccupation with maintaining the international flavor of the ISN, and his consistent insistence on the needs of our colleagues in emerging nations. When taken in the context of the contributions described in this note, these are no mean accomplishments and this Supplement in his honor becomes a small token of our appreciation for these contributions.

Chloride channels are found throughout the nephron. Many chloride channels expressed in the kidney have now been cloned. The CIC family of voltage-dependent chloride channels accounts for most of the known renal chloride channels. Unfortunately, relatively little is known concerning their functional properties. The role that chloride channels may play in renal disease has only recently been addressed.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.

The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.

Cl- channels fused from basolateral mTAL membranes into planar bilayers have distinctive functional characteristics which, when taken together, are unique among Cl- channels. The properties of these 50 to 60 pS channels can account for the characteristics of basolateral Cl- conductances in microperfused mTAL segments and thus may mediate net basolateral Cl- absorption in the intact mTAL. In the present studies, we solubilized basolateral membranes from rabbit mTAL. Since basolateral mTAL Cl- channels contain arginine- and lysine-rich domains, we exposed these solubilized membranes to sequential cation- and anion-exchange chromatography. The bound and unbound eluates from cation- and anion-exchange chromatography were reconstituted into proteoliposomes which, when fused into bilayers, yielded Cl- channels whose properties were virtually identical to those described above for native basolateral mTAL channels fused into bilayers. As judged by valinomycin-sensitive conductive 36Cl- uptake, proteoliposomes reconstituted from the unbound eluates after anion-exchange chromatography were enriched at least 30-fold in Cl- channel activity and had about 30% of the total Cl- channel activity solubilized in native vesicles.

A unique property of basolateral membrane Cl- channels from the mTAL is that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (Po). The K1/2 for maximal activation of Po by Cl- facing intracellular domains of these channels is 10 mM Cl-. The present experiments evaluated the nature of these Cl(-)-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment Po with a K1/2 in the range of 10 mM isethionate without affecting conductance (gCl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mM phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mM), prevented the activation of Po usually seen when the Cl- concentration of solutions facing intracellular channel domains was raised from 2 to 50 mM. However, when the Cl- channel activity was increased by first raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mM, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl- channel faces had no effect on Po. We conclude that the intracellular aspects of these Cl- channels contain Cl(-)-interactive loci (termed [Cl]i) which are accessible to impermeant anions in intracellular fluids and which contain arginine- and lysine-rich domains which can be inactivated, at low ambient Cl- or isethionate concentrations, by interactions with PGO or TNBS.

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8-3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.

Cl- channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl- activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl- in the absence of PKA. The PKA requirement for activation of Cl- channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl- concentrations on the properties of Cl- channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl- channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl- concentration from 2 to 50 mM increased both channel activity (Po) and channel conductance (gCl, pS). Likewise, trans PKA increased mTALH Cl- channel activity by increasing the activity of individual channels when the trans solutions were 2 mM Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect gCl. Moreover, when mTALH Cl- channels were inactivated by reducing cis Cl- concentrations to 50 mM, cis PKA addition did not affect Po. These results are consistent with the view that these Cl- channels originated from basolateral membranes of the mTALH. Cl- channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl- concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl- channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl- channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl- channel activity by both intracellular Cl- and by PKA differ in basolateral mTALH Cl- channels compared to apical Cl- channels from either the tracheal or small intestine.

We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl- concentrations in modulating the activity of Cl- channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, the cis and trans solutions face the extracellular and intracellular aspects of these Cl- channels, respectively. Raising the trans Cl- concentration from 2 to 50 mM increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (delta G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990. J. Membrane Biol. 118:269-278). With 2 mM trans KCl, trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition to cis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50 mM) trans Cl- concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl- channel activity by phosphorylation at or near the Cl(-)-sensitive site on the intracellular face of these channels.

We evaluated the effects of varying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for channel activity, and the effects of varying Cl- concentrations and/or PGO on the relation between holding voltage (VH, mV) and open-time probability (Po). Reducing cis Cl- concentrations, in the range 50-320 mM, produced a linear reduction in fractional open time (Po) with a half-maximal reduction in Po at cis Cl- approximately 170 mM. Channel activity was sustained by equimolar replacement of cis Cl- with F-, but not with impermeant isethionate. For trans solutions, the relation between Cl- concentration and Po was negatively cooperative, with 50% reduction in po at 10 mM Cl-. Reducing cis Cl- had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent energy (delta G) for channel opening. Phenylglyoxal (PGO) reduced Z and altered delta G for Cl- channel activity when added to cis, but not trans solutions. Furthermore, in the presence of cis PGO, reducing the cis Cl- concentration had no effect on Z but altered delta G. Thus we propose that cis PGO and cis Cl- concentrations affect separate sites determining channel activity at the extracellular faces of these Cl- channels.

The present studies examined some of the properties of Cl- channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having a PCl/PK ratio of 10 and a unit conductance of 93 pS in symmetric 320 mM KCl. In asymmetric KCl solutions, the I-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl- conductance of isolated medullary thick ascending limb segments. The Cl- channels incorporated into the planar bilayers were asymmetrical: the trans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10 mM, while the cis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50 mM.