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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide- and chloride-sensitive 22Na+ uptake and barium-sensitive, voltage-dependent 86Rb+ influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1 mM ATP, and 50 mM KCl, the barium-sensitive 86Rb+ influx increased from 361 +/- 138 to 528 +/- 120 pM/mg prot.30 sec (P less than 0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 microgram/ml) was also present in the vesicle solutions. The stimulation of 86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH. [ABSTRACT FROM AUTHOR]

Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage, Ve, which attends net Cl- absorption, JnetCl, and to assess how ADH might augment both JnetCl and Ve in the mouse medullary thick ascending limb of Henle microperfused in vitro. Substituting luminal 5 mM Ba++ for 5 mM K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio, Ra/Rbl, and increasing luminal K+ from 5 to 50 mM in the presence of luminal 10(-4)M furosemide resulted in a 53-mV depolarization of apical membrane voltage, Va. Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH, 10(-4) M luminal furosemide reduced Ve and JnetCl to near zero values and hyperpolarized both Va and Vbl, the voltage across basolateral membranes; however, the depolarization of Vbl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization of Va. Thus ADH-dependent increases in Ve were referable to greater depolarizations of Vbl in the presence of ADH than in the absence of ADH. 68% of the furosemide-induced hyperpolarization of Va was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization of Vbl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl- current. Thus an increase in intracellular Cl- activity may have contributed to the depolarization of Vbl during net Cl- absorption, and the intracellular Cl- activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+, K+, 2Cl- cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+, K+, 2Cl- cotransport units in apical plasma membranes. [ABSTRACT FROM AUTHOR]

This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfused in vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (Gminc) of the transcellular conductance, estimated from Ba++ blockade of apical membrane K+ channels, indicated that Gminc was approximately 30-40% of the measured transepithelial conductance. In apical membranes, K+ was the major conductive species; and ADH increased the magnitude of a Ba++-sensitive K+ conductance under conditions where net Cl- absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl- conductance; this ADH-dependent increase in basal Cl- conductance depended on a simultaneous hormone-dependent increase in the rate of the net Cl- absorption. Cl- removal from luminal solutions had no detectable effect on Ge, and net Cl- absorption was reduced at luminal K+ concentrations less than 5mM; thus apical Cl- entry may have been a Na+, K+, 2Cl- cotransport process having a negligible conductance. The net rate of K+ secretion was approximately 10% of the net rate of Cl- absorption, while the chemical rate of net Cl- absorption was virtually equal to the equivalent short-circuit current. Thus net Cl- absorption was rheogenic; and approximately half of net Na+ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K+ was the principal conductive species in apical plasma membranes, support the view that the majority of K+ efflux from cell to lumen through the Ba++-sensitive apical K+ conductance pathway was recycled into cells by Na+, K+,2Cl- cotransport. [ABSTRACT FROM AUTHOR]