Mamoru Fujimoto

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K+ channel (“Kin channel”) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg2+-and adenosine triphosphate (ATP)-free conditions, where Mg2+-induced inactivation and ATP-induced reactivation of Kin channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, Kin channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg2+ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H+ ionophore, could influence the channel activity reversibly. It is concluded that the activity of Kin channels can be controlled by the intracellular pH under physiological conditions.

Electrochemical Profile for Ion Transport across the Membrane of Proximal Tubular Cells

A micropuncture study was performed on the bullfrog kidney proximal tubular cells utilizing double-barreled ion-selective microelectrodes. The intracellular of Na+, K+, Cl-, HCO3(-) and pH were determined to be 21.6 mEq/L, 67.4 mEq/L, 9.9 mEq/L, 20.2 mEq/L, and 7.49 pH units, respectively. In the extracellular fluid the following activities were found: Na+, 87.4 mEq/L; K+, 2.64 mEq/L; Cl-, 72.5 mEq/L; HCO3(-), 17.9 mEq/L; and pH, 7.66. The membrane potential difference was 68.4 mV and 60.4 mV across the peritubular and brush borders, respectively. The electrochemical potential differences across the individual borders of the proximal tubule cells were separately calculated by setting the intracellular level of both electrical and chemical potentials at zero for convenience. From these analyses, the following interpretations are made. (1) In the net reabsorption of Na+, luminal Na+ enters the cell along a 95-mV gradient across the luminal border and is pumped out to the interstitium against a 104 mV gradient. In the reabsorption of bicarbonate, an uphill pump of about 69 mV (about 70% of the Na+ entry gradient) must exist on the luminal border, of which about 55 mV (80% of the bicarbonate gradient) is accounted for by the H+ secretory pump. (2) In the net reabsorption of K+, a significant K+ uptake pump must exist on the luminal border in addition to the powerful peritubular Na+-K+ exchange pump. The reabsorption of Cl- by the epithelium may take place in two ways: (a) transmembrane transport involving an uphill step of several millivolts, and (b) paracellular leakage through the tight junction. It is thought that the Na+ pump located on the basolateral border of the proximal tubule cell plays a primary role in the regulation of the movement of other ions and water. The regulatory mechanisms of these substances may involve some electrochemical feedback mechanism that works across the proximal tubular epithelium.

Measurement of Intracellular pH of Bullfrog Skeletal Muscle and Renal Tubular Cells with Double-Barreled Antimony Microelectrodes

A pencil-type antimony microelectrode of double-barreled design with a tip of less than 1 to 2 microns in outside diameter was constructed and used to measure intracellular pH(pHi) on frog sartorius muscle and renal tubular cells. Simultaneous observations of membrane potential difference (EM) were made. The results obtained were as follows: (1) The in vivo pHi of frog sartorius muscle was 7.12 +/- 0.07 (SD) (n = 144); the simultaneously measured EM was -51.1 +/- 7.9 mV. The in vivo pHi of frog proximal tubule was 7.49 +/- 0.07 (n = 221) and the EM Peri across the peritubular membrane was -50.2 +/- 9.0 mV. (2) In proximal tubule in vivo, there was a negative correlation between pHi and EM (r = -.62, p < .05). On the other hand, in sartorius muscle in vivo, a positive correlation between the two was found (r = .85, p < .001). (3) In in vitro sartorius muscle, the pHi was 7.03 +/- 0.14 (n = 9) and EM was -62.4 +/- 4.4 mV within one hour after isolation. (4) Increasing the external potassium concentration in the preparations to 75 mM caused a progressive depolarization by 43.3 +/- 15.9 (m = 4) mV, while pHi changed in the alkaline direction by 0.22 +/- 0.03 pH unit. (5) These results indicate that the pHi in both tissues does not obey the Donnan rule.

Reciprocal effects of Ca2+ and Mg-ATP on the ‘run-down’ of the K+ channels in opossum kidney cells

Using the patch clamp technique, we identified an inwardly rectifying K+ channel in the membrane of opossum kidney cells. The single channel conductance was about 90 pS for inward currents and 30 pS for outward currents under a symmetrical high-K+ condition. The activity of the channel was found to decrease with time during recording from inside-out patches. In the solution with submicromolar Ca2+, the activity disappeared within 4–20 min. Intracellular Ca2+ promoted the run-down of the channel activity at 0.1–1 mM, whereas millimolar Mg-ATP restored the activity after run-down. The run-down channels could never be reactivated by ATP in the absence of Mg2+, or by a nonhydrolyzable ATP analog, AMPPNP, even in the presence of Mg2+.

The measurement of intracellular sodium activities in the bullfrog by means of double-barreled sodium liquid ion-exchanger microelectrodes.

A double-barreled Na+-selective microelectrode was constructed with monensin as a liquid ion exchanger. The HCl-treated monensin was dissolved in a solvent (Corning 477317) at 10% (weight/weight). Internal reference solution of its ionic barrel was mixture of 0.49 M NaCl and 0.01 M KCl, the pH being adjusted to 3 with 0.1 M citrate-HCl buffer, whereas that of the PD barrel was 0.5 M KCl. Average slope and selectivity ratio (Na+/K+) tested on 10 different microelectrodes were -57.5 +/- 1.87 mV/P(Na) (SEM) and 6.7 +/- 0.44, respectively. The electrical resistance was an order of 10(10) ohm and the response time was less than 10 sec. Using this microelectrode, a free flow micropuncture experiment was carried out in the bullfrog kidney and the intracellular Na+ activity as well as the membrane PD was determined on the proximal tubular cell. Average value (+/- SEM, n = 15) for the intracellular Na+ and K+ was 20.7 +/- 1.56 mEq/L and 61.2 +/- 1.16 mEq/L, respectively, and -68.7 +/- 0.88 mV for the peritubular membrane PD. There was a significant negative correlation between Na+ and K+ activities within the cell, i.e., the lower the ionic activity of cellular Na+ was, the higher the cellular K+, and vice versa, the sum of these two being kept nearly constant. The above finding may be somehow related to the isosmosis in the reabsorptive process across the proximal tubular epithelium.

THE DOUBLE-BARRELED MICROELECTRODE FOR THE MEASUREMENT OF INTRACELLULAR pH, USING LIQUID ION-EXCHANGER, AND ITS BIOLOGICAL APPLICATION

Publisher Summary In order to determine the intracellular pH (pHi), a double-barreled liquid ion-exchanger (LIX) pH-microelectrode is constructed with nigericin and the tip is made less than 1 μm in outside diameter. This chapter illustrates the construction of the electrode. The electromotive force (EMF) of the LIX pH microelectrode shows a linear response to pH of test solutions within the range of pH 5–9. Various kinds of buffer solutions, such as citrate, phosphate, tris, and borate, are adequate for pH calibrations for this electrode. A couple of coaxial fiber-built-in capillaries are mounted in parallel on a micropipette-puller of horizontal type, rotated 180° under heated platinum ribbon, and pulled to a tip of less than 1 μm outside diameter. The chapter examines the effect of changes in external CO2 on the pH on a single cell of perfused proximal tubule. It also describes the method of the perfused kidney preparation.

Chapter 4 The Direct Measurement of K, Cl, Na, and H Ions in Bullfrog Tubule Cells

Publisher Summary This chapter describes the measurement of some of the intracellular ionic activities of Na + , K + , Cl - , HCO 3 - , and pH in the tubular lumen, cell, and surrounding interstitium in the proximal tubule of the bullfrog ( Rana catesbeiana ), which were obtained with three types of double-barreled ion-selective microelectrodes—namely, parallel, pencil, and tube. The chapter concludes that from an electrochemical point of view, the largest energy dissipation occurs when the luminal Na + enters the cell across the luminal border. To maintain cellular homeostasis, there must be an Na + extrusion pump in exchange for active K + uptake on the peritubular border. From an energetic standpoint, it seems that there is an HCO 3 - uptake pump or H + secretory pump and a K + uptake pump in the luminal border, all of these being somehow related to the downhill movement of Na + . Thus, as to the movement of the Cl - ion, at least in bullfrogs, it seems to distribute passively across the membrane as a balancing ion for Na + , H + , HCO 3 - , or K + along the favorable gradient, which has primarily been generated by active ion movements.

DIRECT MEASUREMENT OF INTRACELLULAR Na AND K ACTIVITIES IN THE RENAL TUBULAR CELLS WITH TRIPLE-BARRELED MICRO-ELECTRODES

Publisher Summary This chapter describes basic characteristics of the triple-barreled microelectrode and presents the data of the animal experiments using this electrode. It discusses that earlier techniques used doublebarreled microelectrode to analyze electrochemical profiles for several kinds of ions in the proximal tubule cell of bullfrogs. It reports a triple-barreled microelectrode, which is capable of measuring the ionic activities of both Na+ and K+ and the membrane potential difference (PD). The tip of the triple-barreled assembly is made less than 0.6 μm in outside diameter. Using this electrode, several micropuncture experiments are carried out for analyzing the mechanism of sugar-evoked potential change of the brush border membrane and the electrogenicity of the Na+ pump located on the peritubular membrane in bullfrog proximal tubule. Administration of ouabain from the peritubular and of cyanide from the luminal sides produces similar changes in different manner. The hyperpolarizations occurring in post-depolarization period are demonstrated to be because of an activation of electrogenic Na+-pump on the peritubular membrane.

REABSORPTIVE MECHANISM OF BICARBONATE IONS ACROSS THE LUMINAL MEMBRANE OF PROXIMAL TUBULE

Publisher Summary The bicarbonate–carbonic acid buffer system plays a key role in the stabilization of reaction of the body fluids. It is well-known that the bicarbonate ion is reabsorbed by the proximal tubule of the kidney. But the reabsorptive mechanism of bicarbonate ion across the luminal and peritubular membranes is not clear, because the direct measurement of bicarbonate ions per se has not yet been done. This chapter presents experiments to measure the intracellular bicarbonate activity (HCO3)i and membrane potential difference (PD) of the proximal tubular cells using double-barreled bicarbonate selective microelectrodes. The bicarbonate-selective double-barrled liquid ion-exchanger microelectrodes are fabricated by the method similar to potassium-selective double-barreled microelectrodes. The chapter explains that both the luminal and peritubular membranes are relatively impermeable to bicarbonate ions. It is likely that this ion is unlikely to penetrate the membrane in ionic form, but moves mainly in the molecular form as CO2.

Regulation of K+ Channels in Proximal Tubules: Studies in Opossum Kidney Cells

Using the patch clamp technique, we examined the properties of an inwardly rectifying K+ (Kin) channel, which contributes greatly to an overall K. conductance in opossum kidney (OK) cells. The Kin channel was sensitive to Ba2+ and quinine, less sensitive to tolbutamide, and insensitive to tetraethylammonium (TEA). Experiments with inside-out patches demonstrated that the activity of Kin, channels was regulated by intracellular Mg2+ and ATP. The Kin channel was inactivated by Mg2+ and reactivated by Mg-ATP, via a process requiring hydrolysis of ATP. The inactivation/reactivation of the Kin channel is suggested to be due to a dephosphorylation/phosphorylation of the channel protein. Intracellular pH also influenced the activity of Kin channels. The activity was low at acid pH and high at alkaline pH, presenting a sigmoidal pH-dependence with a half-maximum activation at pH 7.5. The pH-effects could be attributed to a combination of two different processes: one, a simple binding of H+ to the channel protein, and the other, a H+-induced inactivation of the channel, probably due to a dephosphorylation of the channel protein. These results suggest that under physiological conditions the K+ conductance of OK cell membranes is controlled by intracellular Mg2+, ATP, and pH.