Malcolm Hunter

Human Podocytes Possess a Stretch-Sensitive, Ca2+-Activated K+ Channel: Potential Implications for the Control of Glomerular Filtration

Podocytes express many proteins characteristic of smooth muscle, such as actin and myosin. They also express receptors to several vasoactive agents, including acetylcholine and angiotensin II; these phenotypic properties suggest that podocytes are not static entities but may respond to physiologic stimuli. The electrophysiologic properties of a conditionally immortalized human podocyte cell line that expresses the specific podocyte proteins nephrin, podocin, and synaptopodin were examined by patch clamp. Channels that were highly K(+)-selective and had a conductance of 224 +/- 11.5 pS in symmetrical 150 mM K(+) solutions were identified. Channel activity was Ca(2+)- and voltage-dependent, being increased with an increase in Ca(2+) or depolarization, and inhibited by penitrem A. The conductance and voltage- and Ca(2+)-dependence suggest that this is the large-conductance calcium-activated K(+) channel, BK (KCNMA1)-this was supported by reverse transcription-PCR experiments that showed the presence of the BK encoding mRNA, along with expression of KCNMB subunit types 3 and 4. In sections of human glomeruli, immunocytochemistry revealed that BK co-localizes with the podocyte-specific protein nephrin, indicating that these channels are present in native human podocytes. In whole-cell experiments, penitrem A inhibited outward currents to the same extent as tetra-ethyl ammonium (TEA) but did not affect the membrane potential. Channel activity was also increased by applying suction to the patch pipette or by dilution of the bathing medium, indicating that these channels are stretch sensitive. Thus, these channels do not contribute to the resting membrane potential but are activated by a rise in intracellular Ca(2+), membrane depolarization, cell swelling, or membrane stretch. By implication, these results suggest that podocytes may be able to respond to changes in the glomerular capillary pressure and modulate the GFR.

Role of cell volume and protein kinase C in regulation of a Cl‐ conductance in single proximal tubule cells of Rana temporaria.

1. The whole‐cell patch clamp technique was used to investigate Cl‐ currents in single proximal tubule cells isolated from kidneys of Rana temporaria. 2. Immediately following establishment of the whole‐cell clamp, the Cl‐ conductance (gCl) of the cells was low. However, with 2 mM ATP in the pipette there was a time‐dependent activation of gCl. Such activation was inhibited when the bath contained a hypertonic Ringer solution. 3. The Cl‐ conductance was not directly dependent on cell volume; gCl increased with hypotonic shock and decreased with hypertonic shock, but only in the presence of ATP. 4. Activation of gCl by ATP was dependent on extracellular Ca2+; however, the conductance was not directly Ca2+ sensitive. Activation was inhibited by Gd3+, which also had a direct inhibitory action on gCl. 5. Inhibition of protein kinase C (PKC), by 10 microM PKC pseudo‐substrate (PKC‐ps), completely abolished the ATP‐dependent activation of gCl, while stimulation of PKC, by the PKC activator 4 beta‐phorbol 12‐myristate, 13‐acetate (PMA), increased the degree of activation typically observed with ATP. 6. We propose that gCl is activated by PKC‐mediated phosphorylation and plays a role in volume regulation of the cells.

Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney

Epithelial cells are capable of regulating their volume in response to osmotic swelling or shrinkage. In the present paper a channel is described which may be involved in such a volume-regulatory response. Channels were studied in cell-attached patches of the basolateral membrane of cells isolated from frog kidneys using the patch-clamp technique. The open probability of the channels is increased by the application of negative pressure to the rear of the patch pipette or by bathing the cells in hypotonic fluid. In addition, the channels are voltagesensitive, such that depolarisation increases the open probability. The channels have a conductance of 25 pS with amphibian Ringer as the pipette solution and appear not to discriminate between potassium and sodium. Replacement of chloride by gluconate as the dominant anion in the pipette solution did not affect the current/voltage relationship, suggesting that the channels are cation-nonselective. Inward currents are observed at the resting membrane potential with either potassium or sodium as the dominant cation in the pipette solution: this obviates the channels serving a role as the route for solute exit from the cell during a volume-regulatory decrease response and suggests that they may act as the transduction mechanism sensing changes in cell volume.

Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis.

Decreased sodium (Na+), chloride (Cl−), and water absorption, and increased potassium (K+) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na+ and Cl− absorption are becoming clearer, but the mechanism of increased K+ secretion is unknown. Human colon is normally a K+ secretory epithelium, making it likely that K+ channels are expressed in the luminal (apical) membrane. Based on the assumption that these K+ channels resembled the high conductance luminal K+ (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel α‐subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel α‐subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel α‐subunit protein and mRNA expression (evaluated by western blotting and real‐time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible β‐subunits (β1–4), the β1‐ and β3‐subunits were dominant. Voltage‐dependent, barium‐inhibitable, luminal K+ channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K+ losses in ulcerative colitis, and possibly other diseases associated with altered colonic K+ transport, may reflect wider expression of luminal BK channels along the crypt axis. Copyright

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage‐gated potassium channels are six‐transmembrane (S1–S6) proteins that form a central pore domain (4 × S5–S6) surrounded by four voltage sensor domains (S1–S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper‐phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b–S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization—this motion is accompanied by a reorientation of S4 charges to the extracellular phase.

Non-genomic regulation of intermediate conductance potassium channels by aldosterone in human colonic crypt cells

Background: Aldosterone has a rapid, non-genomic, inhibitory effect on macroscopic basolateral K+ conductance in the human colon, reducing its capacity for Cl− secretion. The molecular identity of the K+ channels constituting this aldosterone inhibitable K+ conductance is unclear. Aim: To characterise the K+ channel inhibited by aldosterone present in the basolateral membrane of human colonic crypt cells. Methods: Crypts were isolated from biopsies of healthy sigmoid colon obtained during colonoscopy. The effect of aldosterone on basolateral K+ channels, and the possible involvement of Na+:H+ exchange, were studied by patch clamp techniques. Total RNA from isolated crypts was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific to intermediate conductance K+ channels (KCNN4) previously identified in other human tissues. Results: In cell attached patches, 1 nmol/l aldosterone significantly decreased the activity of intermediate conductance (27 pS) K+ channels by 31%, 53%, and 54% after 1, 5 and 10, minutes, respectively. Increasing aldosterone concentration to 10 nmol/l produced a further 56% decrease in channel activity after five minutes. Aldosterone 1–10 nmol/l had no effect on channel activity in the presence of 20 μmol/l ethylisopropylamiloride, an inhibitor of Na+:H+ exchange. RT-PCR identified KCNN4 mRNA, which is likely to encode the 27 pS K+ channel inhibited by aldosterone. Conclusion: Intermediate conductance K+ channels (KCNN4) present in the basolateral membranes of human colonic crypt cells are a target for the non-genomic inhibitory effect of aldosterone, which involves stimulation of Na+:H+ exchange, thereby reducing the capacity of the colon for Cl− secretion.

Volume-activated, gadolinium-sensitive whole-cell currents in single proximal cells of frog kidney.

Stretch-activated channels (SACs) have been implicated in the control of epithelial cell volume. Such channels are generally sensitive to the trivalent lanthanide, gadolinium (Gd3+). In this study, using Gd3+ sensitivity and volume activation as indices, we have looked for ionic currents attributable to SACs using the wholecell-patch clamp technique in freshly isolated proximal tubule cells of the frog. Hypotonic shock caused a reversible increase in whole-cell conductance, which was inhibited by Gd3+. In conjunction with this increase in conductance, cell length (measured using an optical technique) also increased. We observed two types of volume and Gd3+-sensitive currents: voltage-dependentIVD and voltage-independent IVI. IVD was found in all cells, activated by depolarisation and hypotonic shock, and was inhibited reversibly by 10 μM Gd3+. The conductance did not discriminate between Na+ and K+ but was slightly anion-selective and was Ca2+-permeable.IVI was observed in only 50% of cells and was also inhibited by Gd3+. Although the inhibition was irreversible, it was dose-dependent, suggesting a specific effect of Gd3+ onIVI. Cells that showed IVI had a significantly higher conductance than those that did not (38.7±4.4,n=20, and 20.5±0.7,n=15, μS · cm−2 respectively). In contrast toIVD,IVI was mildly cation-selective, Ca2+-permeable, and also selective for Na+ over K+. As withIVD, volume-induced increases inIVI were inhibited by Gd3+. Both of these currents are activated during hypotonic shock and may be involved in volume-regulatory processes in frog proximal cells.

Volume regulatory responses in frog isolated proximal cells

Cells respond to increases in volume by activating solute efflux pathways, resulting in water loss and restoration of the original cell volume. The solute efflux pathways underlying these volume regulatory decrease (VRD) responses have been relatively well studied. However, the transduction pathways whereby the change in cell volume is converted into an intracellular signal resulting in VRD are much less well understood. We have examined VRD in isolated proximal tubule cells from the frog, with particular attention to the roles of stretch-activated channels, Ca2+ and protein kinases. Cell length was taken as an index of cell volume, and was measured continuously using a photodiode array. VRD was observed in approximately 50% of cells, and was inhibited by Ba2+, Gd3+ and 4,4′-diisothiocyanatostilbene 2,2′-disulphonic acid (DIDS), and removal of extracellular Ca2+. VRD was accelerated by the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the phosphatase inhibitor F−; on the other hand, VRD was prolonged by 4α-phorbol 12,13-didecanoate (PDC), an inactive phorbol ester), and inhibited by PMA and Gd3+, PMA and 0 Ca2+, and staurosporine. Volume regulation was unaffected by di-butyryl cAMP and 3-isobutyl-1-methyl-xanthene (IBMX). These data suggest that Ca2+ and PKC, via protein phosphorylation, play a stimulatory role in VRD.

Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis.

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na+ and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na+ absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na+ absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface‐crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K+ conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na+ transport, thereby impairing Na+ absorption (and as a consequence, Cl− and water absorption) across the inflamed mucosa. Copyright

Potassium-selective channels in the basolateral membrane of single proximal tubule cells of frog kidney

The membrane potential of proximal tubule cells is dominated by the potassium conductance of the basolateral membrane. In the present paper the nature of this conductance is investigated by the patch-clamp technique. Only one type of K channel was found in the basolateral membranes of freshly isolated proximal cells. In cell-attached patches, the current/voltage relationship is markedly non-linear with much larger inward (30 pS) than outward (≈ 6 pS) conductances, even in the presence of roughly symmetrical K concentrations. Thus the channels show inward rectification. The determination of the conductance for outward current flow is complicated since the current/voltage curves show an area of negative conductance. Nevertheless, taking the conductance for outward current flow and the density of the channels it is possible to account for all of the previously reported potassium conductance of amphibian proximal tubule cells. The open probability of the channels was found not to depend upon the membrane potential. However, the non-linearity of the current/voltage relationships will confer upon the channel the same voltage dependence as that seen in intact proximal tubules, i.e. the conductance decreases with depolarisation. Incubation of cells in Ringer with no substrates or in the presence of alanine and/or glucose showed no change in the activity of the channels. These findings suggest that, although these channels may represent the basolateral conductance of frog proximal tubule cells, they are not involved in the well-established coupling between transport rate and potassium conductance.