We used the patch clamp technique to study the nature of the late sodium current in guinea pig ventricular myocytes. In a cell attached mode of single channel recording at room temperature (22-24 degrees C) two kinds of late (100 msec or more after beginning of the depolarizing pulse) sodium channel activities were recognized. One is isolated brief openings appearing once for about 120 depolarizations per channel (background type), while the other type is sustained openings with rapid interruptions (burst type) that occurred only once for 2,700 depolarizations per channel. The time constant obtained from the open time histogram of the burst type (1.05 msec) was about five times longer than that of background type (0.18 msec, measured at the potential 10 mV above the threshold). Magnitude of the late sodium current flowing through the entire surface of a myocyte was estimated with tetrodotoxin (60 microM), a specific inhibitor of sodium channels, in whole-cell clamp experiments. The steady tetrodotoxin-sensitive current of 12 to 50 pA was registered at -40 mV (26 +/- 14 pA, mean +/- SD, n = 5), in good agreement with the late sodium current calculated from the single channel recording. Tetrodotoxin produced small (congruent to 10%) but significant decreases in the action potential duration. These results suggest the presence of a small but significant late sodium current with slow inactivation kinetics and that this current probably plays a significant role in maintaining the action potential plateau and the duration in guinea pig ventricular myocytes.

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6±6.7 pS (mean±S.D., n=16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33±0.05 (n=10) at the resting potential (RP=-81.7±1.7 mV, n=16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178±25 msec (n=6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0±2.0 msec and 1.92±0.52 sec (n=6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time-and voltage-dependent kinetics.

SUMMARY1. We studied the effects of low temperature on the action potentials and membrane currents of guinea-pig ventricular myocytes, using a tight-seal whole-cell clamp technique.2. The action potential duration at 95 % repolarization was prolonged from 146+ 33 ms (mean+ S.D., n = 6) at 33-34°C (control temperature) to 314+ 83 ms at 24-25°C (low temperature).3. In whole-cell clamp experiments, low temperature decreased the calcium current (ICa), the delayed rectifier potassium current (IK)' and the inwardly rectifying potassium current (IK.) with 'apparent' Qlo (temperature coefficient) values of 2-3 + 06 for ICa' 4-4 + 1-2 for 'K tail current and 1-5 + 03 for IK. (n = 7).4. The effect of low temperature onIK was further studied in the presence of 06 ,(M nicardipine to block ICa. The decay phase of the IK tail consisted of two exponential components. The fast but not the slow component was highly sensitive to the temperature change with an apparent Q1o of 4-5.5. We found that a component of time-independent current is also sensitive to the temperature. The current had a linear I-V relationship and remained almost unchanged after inhibition of Na+-K+ pump in K+-free external solution.6. Using our mathematical model of the ventricular action potential (a modification from the DiFrancesco-Noble model), we simulated the action potential at low temperature by modifying some of the membrane currents, namely IK' IK1, ICa and a component of background current. It was shown that simultaneous changes in these currents could reproduce approximately 75 % of the action prolongation induced by low temperature.

Ionic mechanisms related to the prolongation of cardiac action potential in rats with chronic diabetes mellitus were studied using whole cell voltage-clamp techniques. Diabetes was induced by injection of streptozotocin (STZ; 65 mg/kg body wt) into the tail vein, and ventricular myocytes were isolated from STZ-injected rats (24-30 wk) and from age-matched normal rats. The current densities of transient outward current (Ito), a steady-state outward current, and L-type Ca2+ current (ICa) were significantly smaller in cells from diabetic animals. In addition, the kinetics of Ito of diabetic cells were modified. 1) The decay of Ito was well fitted by a sum of two exponential components in normal cells; there was only one (slow) component in the diabetic cells. 2) The steady-state inactivation curve of Ito in diabetic cells shifted by 5 mV in the negative direction. 3) Recovery from inactivation of Ito was slower in cells from diabetic animals. These alterations in Ito and the steady-state outward current can account for most of the action potential prolongation heretofore documented. The decrease of ICa may possibly be related to the depressed contraction seen in chronic diabetic mellitus.

We examined the effects of oxygen free radicals (OFRs) on action potentials and membrane currents of guinea pig ventricular myocytes. OFRs produced biphasic changes in the action potential duration, initial lengthening (30 s after exposure to OFRs) and subsequent shortening (within 5 min). In voltage-clamp experiments, OFRs suppressed the L-type calcium current, the delayed rectifier K+ current, and the inward rectifier K+ current. In addition, OFRs increased the time-independent outward current (I(term)) at potentials greater than -30 mV. The increases in I(term) reflected activation of the ATP-sensitive K+ (KATP) channels, as glibenclamide (1 microM) blocked this current. In inside-out patches, OFRs significantly increased the open probability of the channel at a relatively narrow range of ATP concentrations (0.2-2 mM), and this effect was enhanced in the presence of ADP (0.1 mM) and abolished in the presence of either free radical scavengers or gliben-clamide. These findings are compatible with the notion that OFRs activate KATP channels by modulating ATP binding sites of the KATP channels, without affecting ADP binding or glibenclamide binding sites.