Basolateral K⁺conductance in principal cells of rat CCD

Abstract

Whole cell K ϩ current was measured by forming seals on the luminal membrane of principal cells in split-open rat cortical collecting ducts. The mean inward, Ba 2ϩ -sensitive conductance, with 40 mM extracellular K ϩ , was 76 Ϯ 12 and 141 Ϯ 22 nS/cell for animals on control and high-K ϩ diets, respectively. The apical contribution to this was estimated to be 3 and 16 nS/cell on control and high-K ϩ diets, respectively. To isolate the basolateral component of whole cell current, we blocked ROMK channels with either tertiapin-Q or intracellular acidification to pH 6.6. The current was weakly inward rectifying when bath K ϩ was Ն40 mM but became more strongly rectified when bath K ϩ was lowered into the physiological range. Including 1 mM spermine in the pipette moderately increased rectification, but most of the outward current remained. The K ϩ current did not require intracellular Ca 2ϩ and was not inhibited by 3 mM ATP in the pipette. The negative log of the acidic dissociation constant (pKa) was ϳ6. homeostasis by setting the resting potential of the cell. K ϩ flux across the basolateral membrane can be in either direction depending on the physiological setting (22,33). In states where K ϩ conservation is required, for example on a low-K ϩ diet or during neonatal growth, K ϩ entering the cell by the active Na ϩ -K ϩ -ATPase is recycled across the basolateral membrane to prevent losses through apical K ϩ channels. In states of K ϩ excess or in the setting of increased aldosterone, the direction of net K ϩ flux across the basolateral membrane reverses (22, 33) such that K ϩ now enters the cell through the basolateral channels and is then secreted into the urine across the apical membrane. Finally, the basolateral K ϩ conductance influences Na ϩ reabsorption by helping to establish the apical membrane potential.Despite its importance, our knowledge of the molecular determinants that underlie the basolateral K ϩ conductance in principal cells is limited. From perfusion studies of isolated rat CCDs, it has been shown that the basolateral K ϩ conductance is Ba 2ϩ sensitive but TEA insensitive (35) and that the fractional apical resistance with amiloride in the lumen is 0.9 (36). The permeability sequence is K ϩ Ͼ Rb ϩ Ͼ NH 4 ϩ (35). Singlechannel recordings from K ϩ channels on the basolateral membrane are technically challenging as the basement membrane presents a barrier to formation of giga-ohm seals on the basolateral surface of these cells. Two approaches have been used to circumvent this problem: enzymatic digestion of the basement membrane (11) and mechanical removal of the adjacent intercalated cells permitting access to the lateral principal cell membrane (50). After collagenase digestion of the basement membrane, a 148-pS channel (cell attached) that decreased to 85 pS when excised into a low-K ϩ bath and a 67-pS channel (cell attached) that decreased to 28 pS when excised were observed (12). The larger conductance channel was inhibited by intracellular acidification with a negative log of the acidic disso...

Quantification of K+ secretion through apical low-conductance K channels in the CCD

“…K loading increases the density of apical SK channels about fourfold while aldosterone alone does not (20). The basolateral K ϩ conductance is enhanced by about twofold with a high-K diet (11). No comparable measurements are available for the aldosterone-treated condition, but we assume a similar increase as both cases are associated with a significant amplication of basolateral membrane surface area (27,28,31).…”

“…Maximal pump rates were set at 17 nA/mm, slightly higher than those measured under nonstimulated conditions (18), and K Na , the intracellular Na concentration required for half-maximal pump turnover, was assumed to be 10 mM. The basolateral K ϩ conductance under conditions of 5 mM peritubular K ϩ was taken to be 5,800 nS/mm (11). Paracellular conductance was assumed to be 300 nS/mm based on a resistance of 1 k⍀ ⅐cm 2 .…”

Kir4.1/Kir5.1 channel forms the major K+channel in the basolateral membrane of mouse renal collecting duct principal cells

“…CCDs were microdissected as previously described (26,32), and principal cells were optically identified as flat and polygonal cells in contrast to the round and protuberant intercalated cells (5,26).…”

“…From whole cell clamp studies on mouse CCD cells (21), CCD-IRK3, a 15-pS inwardly rectifying K ϩ channel cloned from the M1 mouse collecting duct cell line, which presents a high degree of homology with human inwardly rectifying K ϩ (Kir) 2.3 channel (46), is thought to be the 18-pS channel described in rat CCD principal cells (45). Using whole cell patch-clamp technique and noise analysis of K ϩ current measurements of rat CCD principal cells, Gray et al (5) indicated that the native ϳ30-pS K ϩ channel (45) shared some properties of members of the Kir4/Kir5 subfamily of channels. However, no comparison of the properties at the single-channel level of native K ϩ channels in the basolateral membrane of CCD principal cells with those of cloned channels has been conducted.…”

Arachidonic acid inhibits basolateral K channels in the cortical collecting duct via cytochrome P-450 epoxygenase-dependent metabolic pathways

“…Thus it is possible that the 18-pS K channel is a major type of K channel in the basolateral membrane of principal cell in the CCD from the rat kidney. The notion that 18-pS K channels may be major contributors to basolateral K conductance has also been shown with whole cell patch experiments in which noise analysis reveals that 18-pS K channels may account for 65% of whole cell K current (6). Thus inhibition of 18-pS K channels by AA is expected to have an effect on the basolateral membrane potential.…”