Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Ca2(+)-activated K+ channels from rabbit kidney medullary thick ascending limb cells expressed in Xenopus oocytes.

Ca2(+)-activated K+ channels are present in muscle, nerve, pancreas, macrophages, and renal cells. They are important in such diverse functions as neurotransmitter release, muscle excitability, pancreatic secretion, and cell volume regulation. Although much is known about the biophysics of Ca2(+)-activated K+ channels, the molecular structure, cDNA and amino acid sequences are unknown. We injected size-fractionated mRNA isolated from cultured rabbit kidney medullary thick ascending limb cells in Xenopus oocytes and observed newly expressed K+ currents using two-microelectrode voltage-clamp technique. The expressed K+ currents are Ca2+ dependent and inhibited by charybdotoxin, a specific blocker of Ca2(+)-activated K+ channels. Amplitudes of the current ranged from 30 nA to more than 1 microA at a membrane potential of +30 mV. Reversal potential of the current suggested a K(+)-selective channel. The peak activity of Ca2(+)-activated K+ channels were observed in fractions corresponding to a message RNA with size of approximately 4.5 kilobases.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.     

Intercellular fibrillar skeleton in the basal interdigitations of kidney tubular cells

Summary The tubular cells from the thick ascending limb of the loop of Henle in rabbit kidney medulla contain in their basallateral surfaces a complex system of interdigitations. Within these interdigitations, the plasma membranes are separated by extracellular spaces of relatively constant width that contain a previously undescribed fibrillar system. The structural organization and distribution of this intercellular fibrillar skeleton was studied using freeze-fracture etch and then section electron microscopy. The skeleton is comprised of discrete strands with a density of 300 to 400 per m² evenly distributed along the entire basal-lateral region. Each strand has the shape of a brace and it is constructed from up to eight finer filaments each having a width of about 2 nm. The filaments are tightly joined together along their shafts for about 30 nm but they separate at both ends for about 10 nm before contacting the external surface of the plasma membrane. We propose that this intercellular fibrillar skeleton is responsible for maintaining the wide (about 50 nm) and uniform plasma membrane separation along the entire length of the basallateral region of the tubular cells of the thick ascending limb.     

Stimulation of Ca2+-sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney

We used the patch-clamp technique to study the effect of changing the external Ca2+ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2+ concentration from 1 mM to 2 or 3 mM inhibited the basolateral 50-pS K channels while decreasing external Ca2+ to 10 μM ?increased the 50-pS K channel activity. The effect of the external Ca2+ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2+ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2+-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2+ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analog increased the 50-pS K channel activity but did not block the effect of raising the external Ca2+ on the K channels. Neither inhibition of phospholipase A2 (PLA2) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2+ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2+. We conclude that the external Ca2+ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2+ is mediated by the CaSR which stimulates PLC-PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2+ level modulates the reabsorption of divalent cations.     

Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells.

The medullary thick ascending limb of Henle's loop (mTALH) contributes importantly to the regulation of extracellular fluid volume and composition and metabolizes arachidonic acid (AA) chiefly by a cytochrome P450 monooxygenase pathway. Rabbit mTALH cells, when incubated with radiolabeled [14C]AA, form products that segregate into two peaks designated P1 and P2 based on their reverse-phase high pressure liquid chromatography retention times. We have now definitively identified their chemical structures. mTALH cells, isolated from the rabbit outer medulla, were homogenized and incubated with [14C]AA in the presence of NADPH. The AA metabolites in P1 and P2 were identified by gas chromatographic-mass spectrometric methods, including fast atom bombardment, negative ion electron capture, and electron ionization. All mass spectrometric data, the lack of UV chromophores, and comparisons with authentic standards were consistent with P1 containing two principal components: 19-hydroxy-5,8,11,14 eicosatetraenoic acid (19-HETE) and 20 - hydroxy - 5,8,11,14 - eicosatetraenoic acid (20-HETE), P2 contained primarily 1,20-eicosa-5,8,11,14-tetraenedioic acid (20-COOH-AA). The biological properties of P1 and P2 were compared with those of the authentic standards of 19- and 20-HETE and 20-COOH-AA. P1 dose dependently relaxed precontracted mesenteric arterial rings, as did authentic (19S)- and (19R)-HETE, whereas 20-HETE relaxed at lower and contracted at higher concentrations. As P1 contained a mixture of 19- and 20-HETE, each of these AA metabolites presumably contributed to the vascular relaxation produced by P1. Neither P2 nor 20-COOH-AA exhibited vasoactivity, but each demonstrated a similar potency in inhibiting rabbit medullary Na(+)-K(+)-ATPase activity. As previously reported, P2 was a more potent inhibitor of Na(+)-K(+)-ATPase than P1. The lesser inhibitory activity of P1 presumably reflects the presence of similar amounts of 19-HETE, the least active metabolite, and 20-HETE, which resembles 20-COOH-AA in its capacity to inhibit Na(+)-K(+)-ATPase. Thus, the biological activity of the less polar peak, P1, can be accounted for by 19- and 20-HETE, and that of P2, by 20-COOH-AA.     

Affinity and location of an internal K+ ion binding site in shaker K channels

We have examined the interaction between TEA and K+ ions in the pore of Shaker potassium channels. We found that the ability of external TEA to antagonize block of Shaker channels by internal TEA depended on internal K+ ions. In contrast, this antagonism was independent of external K+ concentrations between 0.2 and 40 mM. The external TEA antagonism of internal TEA block increased linearly with the concentration of internal K+ ions. In addition, block by external TEA was significantly enhanced by increases in the internal K+ concentration. These results suggested that external TEA ions do not directly antagonize internal TEA, but rather promote increased occupancy of an internal K+ site by inhibiting the emptying of that site to the external side of the pore. We found this mechanism to be quantitatively consistent with the results and revealed an intrinsic affinity of the site for K+ ions near 65 mM located approximately 7% into the membrane electric field from the internal end of the pore. We also found that the voltage dependence of block by internal TEA was influenced by internal K+ ions. The TEA site (at 0 internal K+) appeared to sense approximately 5% of the field from the internal end of the pore (essentially colocalized with the internal K+ site). These results lead to a refined picture of the number and location of ion binding sites at the inner end of the pore in Shaker K channels.     

Regulation of angiotensin II receptors in the medullary thick ascending limb

Angiotensin II (Ang II) is an important regulator of the function of medullary thick ascending limb of loop of Henle (MTAL). Recent studies showed that changes in Ang II receptor expression occur and underlie changes in the function of proximal tubules during altered sodium intake. The present experiment was designed to determine (1) whether expression of the type 1 Ang II (AT1) receptor in the MTAL is regulated by altered sodium intake, and (2) the specific pathway(s) mediating sodium-induced AT1 expression in the MTAL. Wistar rats were fed a normal sodium (0.5%, NS), low sodium (0.07%, LS), or high sodium (4%, HS) diet for 2 weeks. Northern blot analysis and radioligand binding showed that in rats fed a normal sodium diet the rank of order for both AT1 mRNA expression and receptor density was outer medulla > cortex > inner medulla. Sodium restriction significantly increased both AT1 mRNA expression and receptor density in the outer medulla. In contrast, neither AT1 mRNA expression nor receptor density in the outer medulla was altered by sodium loading. Losartan treatment (3 mg/kg/per day by oral gavage for 2 weeks) prevented low sodium-induced upregulation of the AT1 receptor in the outer medulla, but it had no effect on AT1 expression in the outer medulla of rats fed a normal sodium diet. Highly purified suspensions of MTAL were isolated from rats fed a normal or low sodium diet. Low sodium intake significantly increased AT1 mRNA level by 184% and AT1 receptor density by 58% in MTALs. Primary cultures of MTAL cells were treated with PBS, Ang II (10^-8 M), and Ang II + 17 octadecynoic (17 ODYA, 10 M). Ang II caused about 2-fold increase in AT1 mRNA levels, and this increase was diminished by about 30% by the addition of 17 ODYA. We conclude that (1) sodium restriction but not sodium loading increases AT1 receptor expression in the MTAL, (2) low sodium-induced upregulation of the AT1 receptor in the MTAL is Ang II-dependent, and (3) Ang II-induced upregulation of the AT1 receptor in the MTAL is mediated, at least in part, by cytochrome P450 pathways.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving approximately 37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located approximately 13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.     

Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney

We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20- HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50- 100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.     

Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells

Summary A high-conductance K⁺-selective ion channel was studied in excised membrane patches from human G292 osteoblast-like osteosarcoma cells. Channel conductance averaged 170 pS in symmetric solutions of 153mm KCl, and 135 pS when the pipette was filled with standard saline (150mm NaCl). The probability of the channel being in an open state (P _open) increased with membrane potential, internal calcium, and applied negative pressure. At pCa7, channel activity was observed at membrane potentials greater than 60 mV, while at pCa3, channel activity was seen at 10 mV. Likewise, in the absence of applied pressure, channel openings were rare (P _open = 0.02), whereas with –3 cm Hg applied pressure,P _open increased to 0.40. In each case, i.e., voltage, calcium concentration, and pressure, the increase inP _open resulted from a decrease in the duration of long-closed (interburst) intervals and an increase in the duration of long-open (burst) intervals. Whole-cell responses were consistent with these findings. Hypotonic shock produced an increase in the amplitude and conductance of the outward macroscopic current and a decrease in its rise time, and both single-channel and whole-cell currents were blocked by barium. It is suggested that the voltage-gated, calcium dependent maxi-K⁺ channel in G292 osteoblastic cells is sensitive to membrane stretch and may be directly involved in osmoregulation of these cells. Further, stretch sensitivity o£ the maxi-K⁺ channel in osteotrophic cells may represent an adaptation to stresses associated with mechanical loading of mineralized tissues.     

Lack of inhibition of vasopressin-stimulated NA+K+ATPase by atrial natriuretic factor in the rat renal medullary thick ascending limb of Henle's loop

The anti-diuretic hormone, arginine vasopressin (AVP) stimulates the activity of Na+K+ATPase in the rat renal medullary thick ascending limb of Henle's loop (mTAL). Atrial natriuretic factor (ANF) has been suggested to exert a tubular effect on the mammalian nephron, perhaps in part, by interacting with other hormones. In the present study, we investigated the effect of rat ANF with and without AVP upon mTAL Na+K+ATPase activity using cytochemical methods. ANF alone failed to inhibit or stimulate Na+K+ATPase activity in mTAL at any of the concentrations tested (10 nmol-0.1 pmol l-1). Unlike the rat hypothalamic digitalis-like factor, ANF (10 nmol-10 fmol l-1) did not inhibit Na+K+ATPase activity after stimulation with AVP (1 fmol l-1) for either 4 or 10 min. The results suggest that ANF does not exert an effect on mTAL, either alone or in conjunction with AVP.     

Effect of arachidonic acid on activity of the apical K+ channel in the thick ascending limb of the rat kidney

We have used patch-clamp techniques to study the effects of arachidonic acid (AA) on the activity of the 70-pS K+ channel, the predominant type of the two apical K+ channels operating under physiological conditions in the thick ascending limb (TAL) of the rat kidney. Addition of 5-10 microM AA blocked the activity of the 70-pS K+ channel in both cell- attached and inside-out patches. The inhibitory effect of AA was specific, because application of 10 microM linoleic acid, oleic acid, or palmitic acid failed to mimic the effect of AA. The effect of AA could not be blocked by pretreatment of the TAL tubules with either 5 microM indomethacin (inhibitor of cyclooxygenase) or 4 microM cinnamyl- 3,4-dihydroxy-alpha-cyanocinnamate (CDC) (inhibitor of lipooxygenase). In contrast, addition of 5 microM 17-octadecynoic acid (17-ODYA), an inhibitor of P450 monooxygenases, abolised the effect of AA on the channel activity, indicating that the effect was mediated by cytochrome P450 metabolites of AA. Addition of 10 nM 20-hydroxyeicosatetraenoic acid (20-HETE), the main metabolite of the cytochrome P450 metabolic pathway in the medullary TAL, mimicked the inhibitory effect of 10 microM AA. However, addition of 100 nM 19-HETE or 17-HETE had no significant effects and 100 nM 20-carboxy AA (20-COOH) reduced the channel activity by only 20%, indicating that the inhibitory effect of 20-HETE was specific and responsible for the action of AA. Inhibition of the P450 metabolic pathway by either 5 microM 17-ODYA or 12, 12- dibromododec-11-enoic acid (DBDD) dramatically increased the channel activity by 280% in cell-attached patches. The stimulatory effect of 17- ODYA or DBDD was not observed in inside-out patches. The results strongly indicate that 20-HETE is a specific inhibitor for the 70-pS K+ channel and may play an important role in the regulation of the K+ channel activity in the TAL.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged