Kinetic evidence distinguishing volume‐sensitive chloride current from other types in guinea‐pig ventricular myocytes.

Abstract

1. Kinase-mediated chloride currents (I1c) in guinea-pig ventricular myocytes were activated by application of phorbol ester or forskolin, and compared with currents induced by hyposmotic swelling. Swelling-activated current was identified as Ici from changes in reversal potential, outward rectification and conductance when the Cl-gradient was modified.2. Kinase-stimulated currents were relatively time and voltage independent, whereas hyposmotic swelling-stimulated (hyposmotic-stimulated) currents inactivated during 100 ms puises to positive potentials. Forskolin stimulated time-independent IcI in myocytes with current unresponsive to hyposmotic superfusion, and superimposed a similar pedestal on time-dependent I.c in swollen myocytes.3. Less negative holding potentials depressed hyposmotic-stimulated Ici tested at +80 mV;inhibition was half-maximal at -25 mV. Pulses from -80 to +80 mV inactivated up to 75 % of Ici along a multi-exponential time course; repolarization elicited inwardly developing tail currents whose time courses suggest complex gating. 4. Hyperpolarizations, after strongly-inactivating depolarizations, triggered reactivating tail currents whose amplitude and configuration were dependent on voltage and Cl-gradients; tails were large and inwardly developing at potentials negative to the calculated Clequilibrium potential (Ecl), small and outwardly developing at potentials positive to Ec1, and time independent near Ecl.5. These results suggest that the volume-sensitive Cl-channels investigated here are distinct from other Cl-channels in guinea-pig ventricular myocytes. However, their voltagedependent properties strongly resemble those of volume-sensitive Cl-channels in certain epithelial cells. (Vandenberg et al. 1994), confirmatory biophysical evidence is lacking. The hyposmotic swelling-stimulated ('hyposmoticstimulated') currents recorded in the studies cited above were time independent and had outwardly rectifying current-voltage (I-V) relationships, i.e. they were kinetically indistinguishable from basal (Duan & Nattel, 1994) and kinase-activated (e.g.

A Novel Anionic Inward Rectifier in Native Cardiac Myocytes

“…The SITSsensitive current showed outward rectification and deactivation at more positive potentials ( Figures 5A-d and 5D), which are typical features of I Cl.vol . 10,31,42 The SITS-insensitive inwardly rectifying current ( Figures 5A-c and 5C), however, had properties very similar to those of I Cl.ir observed in mouse cardiac myocytes described earlier in Figure 1. The estimated current densities for guinea pig I Cl.ir in ventricular myocytes (Ϫ10.4Ϯ0.5 pA/pF, at Ϫ120 mV, nϭ3) and atrial myocytes (Ϫ12.8Ϯ0.7 pA/pF, at Ϫ120 mV, nϭ3) revealed no significant differences (PϭNS).…”

“…The TMX-sensitive currents (Figures 6A-e and 6D) were outwardly rectifying and had typical properties of I Cl.vol in the same tissue described previously. 10,31,42 These results suggest that, in addition to the outwardly rectifying I Cl.vol , hypotonic cell swelling also activates an inwardly rectifying I Cl.ir in these guinea pig cardiac myocytes.…”

“…31,41-44 Different from I Cl.ir , however, I Cl.vol is outwardly rectifying, deactivates at positive potentials, has an anion permeability sequence of I Ϫ ϾCl Ϫ , and is sensitive to disulfonic stilbene (such as SITS) Cl Ϫ channel blockers. 31,41,42,44 Figure 5A shows an example of whole-cell currents recorded from a guinea pig ventricular myocyte, in which both I Cl.vol and I Cl.ir were activated under hypotonic conditions. In these experiments, currents were recorded using the same bath and pipette solutions as in Figure 1 and the same voltage-clamp protocol as in Figure 3.…”

Phorbol ester activation of chloride current in guinea‐pig ventricular myocytes

“…Appropriate amounts of stock solutions were added to external and pipette-filling solutions, and corresponding amounts of DMSO ( <0.2%) were also added to the control external solutions. In control experiments (n = 7), further addition of 0.2% DMSO had no (Vandenberg et al, 1994;Shuba et al, 1996), unintentional swelling and turn-on of ICI is a concern in evaluating responses of ICI to pharmacological interventions. However, neither visually-detectable swelling nor activation of 1c, over time occurred in myocytes investigated under isosmotic conditions in the present study.…”

“…Cardiac I1c is stimulated by interventions that cause hyposmotic swelling (Tseng, 1992;Sorota, 1992;Zhang et al, 1993;Vandenberg et al, 1994;Shuba et al, 1996), protein kinase A (PKA) activation (Bahinski et al, 1989;Harvey & Hume, 1989;Ehara & Matsuura, 1993), and protein kinase C (PKC) activation (Walsh, 1991;Walsh & Long, 1994;Zhang et al, 1994). Of these, the regulation by PKC is the least well-characterized (for recent review, see Gadsby et al, 1995).…”