Richard Barrett-Jolley

The Emerging Chondrocyte Channelome

Chondrocytes are the resident cells of articular cartilage and are responsible for synthesizing a range of collagenous and non-collagenous extracellular matrix macromolecules. Whilst chondrocytes exist at low densities in the tissue (1–10% of the total tissue volume in mature cartilage) they are extremely active cells and are capable of responding to a range of mechanical and biochemical stimuli. These responses are necessary for the maintenance of viable cartilage and may be compromised in inflammatory diseases such as arthritis. Although chondrocytes are non-excitable cells their plasma membrane contains a rich complement of ion channels. This diverse channelome appears to be as complex as one might expect to find in excitable cells although, in the case of chondrocytes, their functions are far less well understood. The ion channels so far identified in chondrocytes include potassium channels (KATP, BK, Kv, and SK), sodium channels (epithelial sodium channels, voltage activated sodium channels), transient receptor potential calcium or non-selective cation channels and chloride channels. In this review we describe this emerging channelome and discuss the possible functions of a range of chondrocyte ion channels.

Rapid neuromodulation by cortisol in the rat paraventricular nucleus: an in vitro study

We have used a range of in vitro electrophysiological techniques to investigate the mechanism of rapid cortisol neuromodulation of parvocellular neurones in the rat paraventricular nucleus. In our study, we found that cortisol (10 μM) increased spontaneous action–current firing frequency to 193%. This effect was insensitive to the glucocorticoid intracellular‐receptor antagonist mifepristone. Cortisol (0.1–10 μM) had no detectable effects on whole‐cell GABA current amplitudes, or GABAA single‐channel kinetics. Cortisol (10 μM) inhibited whole‐cell potassium currents in parvocellular neurones by shifting the steady‐state activation curve by 14 mV to the right. Additionally, in a cell line expressing both the glucocorticoid intracellular receptor and recombinant, fast inactivating potassium channels (hKv1.3), cortisol (1 and 10 μM) inhibited potassium currents by shifting their steady‐state activation curves to the right by 12 mV (10 μM cortisol). This effect was also insensitive to the cortisol antagonist, mifepristone. These data suggest that inhibition of voltage‐gated potassium channels may contribute to the rapid neuromodulatory effects of cortisol, possibly by direct interaction with the ion channel itself.

Cell Volume Regulation in Chondrocytes

Chondrocytes are the cells within cartilage which produce and maintain the extracellular matrix. Volume regulation in these cells is vital to their function and occurs in several different physiological and pathological contexts. Firstly, chondrocytes exist within an environment of changing osmolarity and compressive loads. Secondly, in osteoarthritic joint failure, cartilage water content changes and there is a notable increase in chondrocyte apoptosis. Thirdly, endochondral ossification requires chondrocyte swelling in association with hypertrophy. Regulatory volume decrease (RVD) and regulatory volume increase (RVI) have both been observed in articular chondrocytes and this review focuses on the mechanisms identified to account for these. There has been evidence so far to suggest TRPV4 is central to RVD; however other elements of the pathway have not yet been identified. Unlike RVD, RVI appears less robust in articular chondrocytes and there have been fewer mechanistic studies; the primary focus being on the Na+-K+-2Cl- co-transporter. The clinical significance of chondrocyte volume regulation remains unproven. Importantly however, transcript abundances of several ion channels implicated in volume control are changed in chondrocytes from osteoarthritic cartilage. A critical question is whether disturbances of volume regulation mechanisms lead to, result from or are simply coincidental to cartilage damage.

Characterization of a stretch-activated potassium channel in chondrocytes

Chondrocytes possess the capacity to transduce load‐induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch‐clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell‐attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm2). The mean slope conductance of the principal single channels resolved within the total stretch‐activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, EK+, suggesting it was a high‐conductance potassium channel. Activation of these high‐conductance potassium channels was inhibited by extracellular TEA (Kd approx. 900 µM) and iberiotoxin (Kd approx. 40 nM). This suggests that the current was largely carried by BK‐like potassium (MaxiK) channels. To further characterize these BK‐like channels, we used inside‐out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1‐ and β1‐subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction. J. Cell. Physiol. 223: 511–518, 2010.

The role of the membrane potential in chondrocyte volume regulation

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium‐selective and chloride‐selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium‐sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔVm = −30 ± 4 mV). Analysis of the gadolinium‐sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔVm = −18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at −80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979–2986, 2011.

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine

We have investigated the inhibition of inwardly rectifying potassium channels by the α‐adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two‐electrode voltage‐clamp of rat isolated flexor digitorum brevis muscle and whole‐cell patch‐clamp of cell lines transfected with Kir2.1 (IRK1). In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α2‐adrenergic receptors and an antagonist at α1x‐receptors, was found to inhibit the inward rectifier current with a Ki of 30 μM. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α1‐(prazosin) or α2‐(rauwolscine) antagonists. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f. ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp((Vm+149)/22) s. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. The data suggest that the clonidine‐like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of α‐receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

Characterization of KATP channels in intact mammalian skeletal muscle fibres

1 The aim of this study was to characterize the KATP channel of intact rat skeletal muscle (rat flexor digitorum brevis muscle). Changes in membrane currents were recorded with two‐electrode voltage‐clamp of whole fibres. 2 The KATP channel openers, levcromakalim and pinacidil (10–400 μM), caused a concentration‐dependent increase in whole‐cell chord conductance (up to approximately 1.5 mScm−2). The activated current had a weak inwardly rectifying current‐voltage relation, a reversal potential near EK and nanomolar sensitivity to glibenclamide – characteristic of a KATP channel current. Concentration‐effect analysis revealed that levcromakalim and pinacidil were not particularly potent (EC50 ∼186 μM, ∼30 μM, respectively), but diazoxide was completely inactive. 3 The ability of both classical KATP channel inhibitors (glibenclamide, tolbutamide, glipizide and 5‐hydroxydecanoic acid) and a number of structurally related glibenclamide analogues to antagonize the levcromakalim‐induced current was determined. Glibenclamide was the most potent compound with an IC50 of approximately 5 nM. However, the non‐sulphonylurea (but cardioactive) compound 5‐hydroxydecanoic acid was inactive in this preparation. 4 Regression analysis showed that the glibenclamide analogues used have a similar rank order of potency to that observed previously in vascular smooth muscle and cerebral tissue. However, two compounds (glipizide and DK13) were found to have unexpectedly low potency in skeletal muscle. 5 These experiments revealed KATP channels of skeletal muscle to be at least 10× more sensitive to glibenclamide than previously found; this may be because of the requirement for an intact intracellular environment for the full effect of sulphonylureas to be realised. Pharmacologically, KATP channels of mammalian skeletal muscle appear to resemble most closely KATP channels of cardiac myocytes.

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

Abstract. We investigated the block of KATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited KATP channels with an apparent Ki of 63 nm and a Hill coefficient of 0.85. The inhibition of KATP channels by glibenclamide was unaffected by internal Mg2+. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and KD to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 107m−1 sec−1 and an unbinding rate of 6.26 sec−1. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 106m−1 sec−1.

Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+‐sensitive K+ channels in rat mesenteric artery

•  Relaxation of vascular smooth muscle, which increases blood vessel diameter, is often mediated through vasodilator‐induced elevations of intracellular 3′‐5′‐cyclic adenosine monophosphate (cAMP), although the mechanisms are incompletely understood. •  In this study we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. •  We show that Epac mediates vasorelaxation in mesenteric arteries by facilitating the opening of several subtypes of Ca2+‐sensitive K+ channel within the endothelium and on vascular smooth muscle. •  Epac‐mediated hyperpolarization of the smooth muscle membrane brought about by opening of these channels acts to limit Ca2+ entry via voltage‐gated Ca2+ channels leading to vasorelaxation. •  This represents a potentially important, previously uncharacterised mechanism through which vasodilator‐induced elevation of cAMP can regulate vascular tone and thus blood flow.