Trevor Powell

Cell swelling and ion transport pathways in cardiac myocytes

Cell swelling causes stretch and/or deformation of cell membranes and the underlying cytoskeletal network as well as dilution of intracellular contents [l]. It is therefore not surprising that most mammalian cells, including cardiac myocytes, respond to swelling by modulating transporters and or ion channels that permit efflux of intracellular osmolytes (and osmotically obliged water) which will tend to restore cell volume to its original value [2-51. This is of particular significance in the heart as many of the transport pathways modulated by cell swelling are electrogenie, so their modulation will alter excitability of the heart. This is likely to be most important in the context of myocardial ischaemia and reperfusion as this is when cell swelling is most significant [6] and when arrhythmias are most common [7]. Recent work, using a range of techniques including patch-clamp analysis of isolated cardiac myocytes, is beginning to unravel the mechanisms underlying osmotic modulation of electrical activity in the heart and it is this work that will be the primary focus of this review.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

Expression of Ca2+‐permeable two‐pore channels rescues NAADP signalling in TPC‐deficient cells

The second messenger NAADP triggers Ca2+ release from endo‐lysosomes. Although two‐pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP‐dependent Ca2+ responses as assessed by single‐cell Ca2+ imaging or patch‐clamp of single endo‐lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re‐expressing wild‐type TPCs, but not by mutant versions with impaired Ca2+‐permeability, nor by TRPML1. Another mouse line formerly reported as TPC‐null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP‐induced Ca2+ release. High‐affinity [32P]NAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+‐permeable channels indispensable for NAADP signalling.

Extracellular osmotic pressure modulates sodium-calcium exchange in isolated guinea-pig ventricular myocytes.

1. The sensitivity of the cardiac Na(+)‐Ca2+ exchange current to changes in osmotic pressure was investigated in guinea‐pig ventricular myocytes, using the whole‐cell patch‐clamp technique. 2. A hyposmotic challenge applied by removal of sucrose from the standard bathing solution reduced exchanger current, measured as the Ni(2+)‐sensitive component of whole‐cell transsarcolemmal current. These changes were fully reversible. 3. No response of whole‐cell current to hyposmosis was observed when Ca2+ was removed from the bathing solution by chelation with 1 mM EGTA. 4. Inclusion of 25 microM exchanger inhibitory peptide (XIP) in the pipette solution caused a marked reduction in the Ni(2+)‐sensitive component of membrane current, but the percentage change in Ni(2+)‐sensitive membrane slope conductance evoked by hyposmosis was the same as when XIP was omitted from the pipette solution. 5. Exposure of cells to hyperosmotic solutions produced variable responses. In a majority of cells, solutions 30% hyperosmotic compared with control evoked a persistent increase in exchanger current, whereas for solutions 50% hyperosmotic, a larger but transient increase in current was observed. 6. Over a wide range of osmolalities (50‐130% of isosmotic) the changes in Ni(2+)‐sensitive membrane slope conductance were linearly related to the changes in extracellular osmotic pressure. 7. We propose that one consequence of exposing ventricular myocytes to anisosmotic solutions is modulation of Na(+)‐Ca2+ exchange current.

Turnover rate of the cardiac Na(+)‐Ca2+ exchanger in guinea‐pig ventricular myocytes.

1. Single guinea‐pig ventricular myocytes were voltage clamped using the whole‐cell configuration of the patch‐clamp technique and membrane current generated by the Na(+)‐Ca2+ exchange mechanism recorded. 2. Rapid increases in cytosolic free calcium ([Ca2+]i) evoked by flash photolysis of either nitr‐5 or DM‐nitrophen resulted in current relaxations, arising from a redistribution of exchanger carrier conformations induced by the changes in [Ca2+]i. 3. Relaxation time constants were temperature dependent with a temperature coefficient over a 10 degrees C range (Q10) of approximately 3 and also voltage dependent, decreasing on hyperpolarization for membrane potentials in the range +40 to ‐80 mV. 4. The experimental results are consistent with consecutive exchange models having electrogenic Na+ translocation steps, together with a site density and turnover rate similar to that for the Na(+)‐K+ pump.

Electrophysiological Demonstration of Voltage- Activated H+ Channels in Bovine Articular Chondrocytes

Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pHi), so characterising the membrane transport pathways that determine pHi is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltageactivated H+ channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H+, the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pHo) and pHi, and inhibited by Zn2+. The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.

Osmotic shock : modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart

To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer. 31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 +/- 2% and pHi by 0.02 +/- 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 +/- 1% and pHi by 0.14 +/- 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+ exchange with use of 1 microM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury.To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer.31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 ± 2% and pHi by 0.02 ± 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 ± 1% and pHi by 0.14 ± 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+exchange with use of 1 μM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury.

HIGH RESOLUTION SCANNING FORCE MICROSCOPY OF CARDIAC MYOCYTES

The advent of scanning probe microscopy has introduced a powerful new method of probing the structural features of biological specimens. In this study, high resolution atomic force microscopy micrographs of single, isolated, cardiac myocytes are presented. Significantly, our images show not only the features to be expected of the external sarcolemma, but also resolve sub‐surface features, including the striated pattern of the contractile proteins and their associated sarcoplasmic reticulum and mitochondria.