Christopher J. Winters

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

SummaryWe evaluated the effects of vawrying 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 efects of varying Cl− concentrations and/or PGO on the relation between holding voltageVH-mV) and open-time probability (Po).Reducingcis Cl− concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (Pv) with a half-maximal reduction inPo atcis Cl−≈170mM. Channel activity was sustained by equimolar replacement ofcis Cl− with F−, but not with impermeant isethionate. Fortrans solutions, the relation between Cl− concentration andP0 at 10mm Cl−. Reducingcis Cl− had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (δG) for channel opening.Phenylglyoxal (PGO) reducedZ and altered δG for Cl− channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl− concentration had no effect onZ but altered δG. Thus we propose thatcis PGO and,cis Cl− concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl− channels.

Cl− channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl− or cAMP-dependent protein kinase

SummaryWe 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, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl− channels, respectively. Raising thetrans Cl− concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (ΔG) 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 2mmtrans 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 tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)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.

Cl- channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl- channels with apical Cl- channels from jejunum and trachea.

SummaryCl− 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.

Cl− channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

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 (P0). The K1/2 for maximal activation of P0 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 P0 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 P0 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 P0. 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 arginineand lysine-rich domains which can be inactivated, at low ambient Cl− or isethionate concentrations, by interactions with PGO or TNBS.

Cl(-) channels in basolateral TAL membranes XV. Molecular heterogeneity between cortical and medullary channels.

Abstract. 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 Po 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 (T1/2, sec) for 36Cl− 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 T1/2 for 36Cl− 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.

Cl? Channels in Basolateral TAL Membranes. XVII. Kinetic Properties of mcClC-Ka, a Basolateral CTAL Cl? Channel

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.

Chloride Channels in Basolateral TAL Membranes. XVIII. Phenylglyoxal Induces Functional mcClC-Ka Activity in Basolateral MTAL Membranes

Cultured mouse MTAL cells contain more mRNA encoding the Cl− channel mcClC-Ka, which mediates CTAL Cl− absorption, than mRNA encoding the Cl− channel mmClC-Ka, which mediates MTAL Cl− absorption. mmClC-Ka and mcClC-Ka have three functional differences: 1) mmClC-Ka open time probability, Po, increases with increasing cytosolic Cl−, but variations in cytosolic Cl− do not affect Po in mcClC-Ka; 2) mmClC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on Po in mcClC-Ka; and 3) mmClC-Ka channels have single-ion occupancy, while mcClC-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 mcClC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl− access to arginine or lysine residues on mmClC-Ka by PGO results in Cl− channels having the functional characteristics of mcClC-Ka channels.

Cl− Channels in Basolateral TAL Membranes. XIX. Cytosolic Cl− Regulates mmClC-Ka and mcClC-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: 10−6M PGE2 and 10−4M bumetanide. Basolateral Cl− channels from control cultured MTAL cells exhibited the signature characteristics of mmClC-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 10−6M PGE2 or 10−4M bumetanide in culture media reduced steady-state MTAL cytosolic Cl−. Chloride channels from these cells exhibited characteristics unique to CTAL mcClC-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 10−6M PGE2 or 10−4M bumetanide reduced mRNA levels encoding mmClC-Ka but not mRNA levels encoding mcClC-Ka. However, when MTAL cells were cultured under control conditions, and then pre-incubated for 60 minutes with 10−4M bumetanide, cytosolic Cl− fell acutely but Cl− channels exhibited characteristics of mmClC-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 mmClC-Ka synthesis.