A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current‐clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl− concentration and shortened by replacement of extracellular Cl− with I−. Under voltage‐clamp, membrane potential steps or ramps evoked an anionic background current (IAB) carried by either Cl−, Br−, I− or NO3−. Activation of IAB was Ca2+‐ and cyclic AMP‐independent, and was unaffected by cell shrinkage. IAB was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca2+‐, cyclic AMP‐ and swelling‐activated Cl− currents in ventricular cells of other mammals. These results suggest that IAB may be carried by a novel class of Cl− channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that IAB could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl− channels as potential targets for novel antiarrythmic agents.

Introduction Cl 7 currents have been described in atrial and ventricular myocytes isolated from the hearts of many mammalian species and have been implicated in both cardiac physiology and pathophysiology (see reviews by: Ackerman & Clapham, 1993;Faivre & Bril, 1997;Hiraoka et al., 1998;Sorota, 1999). In cardiac myocytes, three main classes of Cl 7 current have been described: (i) a calcium-activated Cl 7 current activated following elevation of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) (Zygmunt & Gibbons, 1992;La¯amme & Becker, 1996); (ii) a swelling-activated current, activated following hypotonic shock (Du & Sorota, 1997;Vandenberg et al., 1997); and (iii) a cyclic AMP-activated current activated following b-adrenergic receptor stimulation (Harvey & Hume, 1989;Bahninsky et al., 1989;Levesque & Hume, 1995). The channel which carries the cyclic AMP-activated current is the product of the cystic ®brosis gene known as the cystic ®brosis transmembrane conductance regulator (CFTR; see Sorota, 1999). Activation of Cl 7 currents can potentially markedly aect generation and propagation of cardiac action potentials since the Cl 7 equilibrium potential (E Cl ) in intact ventricular cells is approximately 750 mV. Thus, at potentials hyperpolarized to E Cl , inward Cl 7 current would induce depolarization and at potentials depolarized to E Cl outward Cl 7 current would induce repolarization. It is this feature of cardiac Cl 7 channels that makes their blockade a potential approach for therapy of cardiac arrhythmia.
It is important to note that Cl 7 channels are generally permeable to other halides and other anions including amino acids such as aspartic acid and taurine in addition to Cl 7 (Hall et al., 1996), and therefore may be regarded as anion channels. We have used this feature of Cl 7 channel physiology to examine the eects of anions on the action potential in rat ventricular myocytes. Our results show that the duration of the rat ventricular action potential is acutely sensitive to extracellular anions. In addition, we have obtained evidence supporting the existence of a novel anionic background current (I AB ) which is not dependent upon the presence of intracellular Ca 2+ , cyclic AMP or cell-swelling.
In electrophysiological experiments, the reference Ag/AgCl electrode was immersed in a solution of 3 M KCl continuous with an agar bridge (4% agar in 3 M KCl) to minimise junction potential changes. Voltage-clamped myocytes were subjected to membrane potential steps from a holding potential of 780 mV at 0.33 Hz and`saw-toothed' ramps from 750 mV. During ramps, cells were depolarized at a rate of 0.32 V s 71 from 790 to +70 mV and back to 790 mV. Membrane currents and potentials were recorded on digital audio tape and subsequently digitized, signal-averaged over ten stimulations and ®ltered at frequencies appropriate to the Nyquist criterion (Stanley et al., 1984). Cell capacitance, for current normalization, was calculated by direct integration of the current transients evoked by 20 mV hyperpolarizing voltage steps of 5 ms duration, applied at 20 Hz. Mean measurements are presented with their respective standard errors, and statistical signi®cance was assessed using the Student tdistribution. When stated in the text,`signi®cance' refers to the 95% level of con®dence (P50.05). Figure 1a is a typical rat ventricular action potential recorded from a cell dialysed with solution E under current-clamp. The mean control action potential duration measured following 90% repolarization (APD 90 ) was 66+11 ms (n=13). Reduction of the extracellular Cl 7 concentration ([Cl 7 ] e ) from 155 to 35 mM in Solution A caused slight hyperpolarization of resting membrane potential from 773+3 mV to 776+3 mV (n=15) and signi®cantly prolonged APD 90 by 13+4 ms (18+3%, n=9). Upon replacement of 94% of the extracellular chloride in solution A with iodide, resting membrane potential underwent a slight depolarization to 770+3 mV (n=9). This was coupled to a signi®cant shortening in APD 90 of 11+2 ms (24+3%, n=7) compared to control (see Figure 1a). These changes in APD persisted after normalization, as shown in Figure 1b, indicating that they were independent of the eects on resting potential. In order to determine the cause of these changes in APD, voltage-clamp experiments were performed under conditions that only allowed anion movement (using Solutions B & D). Shown in Figure 1c are the eects of applying 800 ms membrane potential steps in the presence of a range of extracellular chloride concentrations. The current-voltage (I ± V) curves shown in Figure 1c, together with those from other experiments, allowed the determination of reversal potentials (E rev ) for chloride. The experimentally derived values for E rev were 738+2 mV (n=12), 719+2 mV (n=7) and 717+1 mV (n=6) for 152, 97 and 37 mM chloride, respectively; with the corresponding theoretical reversal potentials being 752, 740 and 717 mV, respectively. Although the reversal potentials for 152 and 97 mM extracellular Cl 7 deviated from the theoretical, the current was acutely sensitive to changes in the concentration of the Special Report anion suggesting it was indeed carried through an anion channel. To verify this notion, 90% of the extracellular Cl 7 in solution B was substituted with I 7 , Br 7 or NO 3 7 . Figure 1d shows current density I ± V curves from these experiments. The outward current density was greater in the presence of I 7 and NO 3 7 than Br 7 (which was similar to Cl 7 ). The aniondependent shifts in E rev in these experiments were used in the modi®ed Goldman-Hodgkin-Katz equation (see Du & Sorota, 1997) to obtain relative permeabilities for these anions through the putative anion channel. Permeabilities of 1.8+0.3 for NO 3 7 (n=4), 1.5+0.3 for I 7 (n=5), and 0.7+0.1 for Br 7 (n=3) were obtained, resulting in the following permeability sequence: NO 3 7 5I 7 4Cl 7 ,5Br 7 . To further explore the eects of NO 3 7 and I 7 , voltage-clamped myocytes held at 750 mV in solution C were subjected to continuous trains of membrane potential ramps. For clarity, Figure 2a shows dierence currents calculated by subtacting the current recorded during negative-going ramps in the presence of the relatively impermeant anion, aspartate 7 . The permeabilities of both NO 3 7 and I 7 , relative to Cl 7 , were, coincidentally, found to be 1.5+0.1 (n=6) similar to the results from voltage-step experiments (above). Currents generated by voltage ramps in the presence of I 7 (solution C) or Cl 7 (solution B) were insensitive to 50 ± 100 mM DIDS (n=7; e.g. Figure 2b), 50 ± 100 mM ni¯umic acid (n=8; e.g. Figure 2c) or 50 mM 9-AC (data not shown), concentrations of blockers known to have marked inhibitory eects on cardiac Cl 7 channels (see Sorota, 1999). These results suggest that the current does not fall into a recognized class of cardiac anion currents. In order to con®rm this notion, the eects of (i) extracellular application of 400 mM IBMX, 10.0 mM FSK and 0.1 mM isoproterenol (contained in solution B) were examined to test for CFTR involvement; (ii) extracellular tonicity was increased to verify whether cell shrinkage aected current amplitude; and (iii) 1 mM EGTA was added to the pipette solution to determine the requirement for intracellular Ca 2+ . Figure 2d shows that ramp currents obtained in the presence of Solution B were virtually unaected by the elevation of the intracellular concentration of cyclic AMP (n=11) sucient to double the amplitude of the L-type Ca 2+ current in the same cells (not shown). Cell shrinkage induced by a 20% increase in the extracellular tonicity using NMDG aspartate was without eect (n=10), as was the addition of 1 mM EGTA to the intracellular solution (not shown), even though cell contractions were abolished. These observations all strongly suggest that the conductance demonstrated here did not fall into any of the three main classes of cardiac Cl 7 channel.

Discussion
Our results demonstrate the existence of, what appears to be, a novel anionic background current (I AB ) in ventricular myocytes isolated from the rat heart: a ®nding with important implications for those interested in the potential value of Cl 7 channels as therapeutic targets. The channel underlying I AB appears to be permeable to a range of anions, but its activation is not dependent upon intracellular Ca 2+ , cyclic AMP or cell-swelling. Alteration of the anionic conditions, which aected the magnitude of I AB had profound eects on action potential duration, suggesting that I AB plays a role in controlling the pro®le of the rat ventricular action potential.
It appears that the permeability sequence for I AB (ie. NO 3 7 5I 7 4Cl 7 5Br 7 ) is similar to that of swelling-activated Cl 7 channels (Sorota, 1997), but, for halides, is the opposite to that of CFTR (Ackermann & Clapham, 1993;Sorota, 1999). However, upon altering the chloride gradient, the reversal potential of I AB deviated slightly from Nernstian theory suggesting that the biophysical characteristics of the channels carrying I AB deviate from ideality. This is perhaps not surprising however, in view of the apparently wide range of anions that support I AB .
Stilbenes and fenamates are extremely promiscuous blockers, aecting a wide range of anion transporters and channels. DIDS and ni¯umic acid at the concentrations used in this study (50 ± 100 mM) abolish swelling-activated and Ca 2+activated I Cl in cardiac cells (see Du & Sorota, 1997;Zygmunt & Gibbons, 1992). CFTR channels are also at-least partially sensitive to ni¯umic acid (Sorota, 1999). Yet these molecules and 9-AC were ineective against I AB . Furthermore, the experiments designed to examine the role of [Ca 2+ ] i , cyclic AMP and cell volume revealed no large changes in the magnitude of I AB . Taken together, these results provide strong evidence that I AB could be carried by a novel class of Cl 7 channel. However, the possibility that I AB channels represent an atypical variant of one of the three main classes of cardiac Cl 7 channel cannot be excluded.
Action potentials were observed to be prolonged by a reduction of [Cl 7 ] e and shortened when I 7 was the predominant extracellular anion. When [Cl 7 ] e was reduced from 155 to 35 mM, the observed E rev for Cl 7 shifted by +21 mV, readily explaining the prolongation of APD. Such a change in Cl 7 gradient would result in inward I AB¯o w during repolarization. This increase in inward Cl 7 current at potentials negative to E Cl would counteract in part, some of the outward currents¯owing, thereby prolonging APD. APD shortening after replacing extracellular Cl 7 by I 7 can be readily explained by the increase in outward I AB carried by I 7 . Superfusion with low [Cl 7 ] e and extracellular I 7 caused hyperpolarization and depolarization of the RMP, respectively. Since an increased inward Cl 7 current at low [Cl 7 ] e would be expected to depolarize the cell, these eects on RMP may be mediated independently of I AB . Nevertheless, upon normalizing the action potential (Figure 1b), it was shown that the eects of anions on APD were independent of those on RMP.
In conclusion, our results are consistent with the existence of a novel background anionic current in rat ventricular muscle (I AB ) which may play a major role in controlling APD. These ®ndings have important implications for those studying cardiac Cl 7 channels as potential targets for novel antiarrythmic agents. It remains to be determined whether I AB is present in human ventricular muscle, although preliminary evidence suggests that a background Cl 7 current may be present in human atria (Berul et al., 1997). Thus, I AB may represent a novel therapeutic target for anti-arrhythmic agents. In any event, in light of this data, caution should be exercised when interpreting the eects of anions or potential antiarrhythmic agents on rat cardiac tissue. R.Z. Kozlowski is a British Heart Foundation Lecturer. This work was funded in part by the Medical Research Council of Great Britain.