Jonathan C. Makielski

Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine.

To investigate possible ionic current mechanisms underlying ischemic arrhythmias, we studied single Na+ channel currents in rat and rabbit cardiac myocytes treated with the ischemic metabolite lysophosphatidylcholine (LPC) using the cell-attached and excised inside-out patch-clamp technique at 22 degrees C. LPC has been reported previously to reduce open probability and to induce sustained open channel activity at depolarized potentials. We now report two new observations for Na+ currents in LPC-treated patches: 1) The activation-voltage relation of the peak of the ensemble currents is shifted in the negative (hyperpolarizing) direction by approximately 20 mV compared with control currents. This effect was observed in all patches for depolarizations from a holding potential of -150 mV to different test potentials. 2) In some LPC-treated patches, Na+ channels exhibited sustained bursting activity at potentials as negative as -150 mV, giving a nondecaying inward current. This bursting activity was accompanied by double and triple simultaneous openings and closings, suggesting tight cooperativity in channel gating. These LPC-modified channels were identified as Na+ channels, because their unitary conductance was the same as Na+ channels in control solutions, because the single channel current-voltage relation was extrapolated to reverse at the Na+ Nernst potential, and because the current was blocked by the local anesthetic QX-222. This novel depolarizing current may play a role in the electrophysiological abnormalities in ischemia, including abnormal automaticity and reentrant arrhythmias, and could be a target for antiarrhythmic drugs.

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.

Spontaneous Coronary Vasospasm in KATP Mutant Mice Arises From a Smooth Muscle–Extrinsic Process

In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle–specific SM22&agr; promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle–extrinsic process.

Block of sodium current by heptanol in voltage-clamped canine cardiac Purkinje cells.

Heptanol blocks sodium current (INa) in nerve, but its effects on cardiac INa have not been well characterized. Block of INa by heptanol was studied in 16 internally perfused voltage-clamped cardiac Purkinje cells at reduced Na+ (45 mM outside, 0 mM inside). Heptanol block of peak sodium conductance was well described by a single-site binding curve with half block at 1.3 mM (20 degrees C) and showed no use dependence. With 1.5 mM heptanol, block increased slightly by 0.7%/degrees C from 10 degrees C to 27 degrees C. With 3.0 mM heptanol, steady-state availability shifted by 9.4 +/- 1.3 mV (n = 6) in the hyperpolarizing direction, and steady-state activation shifted by 8.3 +/- 2.2 mV (n = 5) in the depolarizing direction, thus closing off the INa window current. Heptanol also decreased the time to peak and accelerated the decay of INa. Similar results were found with octanol at lower concentrations. These alcohols have important effects on cardiac INa at concentrations used in studies for cellular uncoupling in heart.

ATP-sensitive K+ channel opener acts as a potent Cl- channel inhibitor in vascular smooth muscle cells

We describe the activation of a K+ current and inhibition of a Cl− current by a cyanoguanidine activator of ATP-sensitive K+ channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nm to 1 μm) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 μm). The U83757induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlylammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nm to 300 μm) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where Ek=−82 mV and ECl=0 mV, the reversal potential of the U83757-sensitive current was approximately −70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl− channel blocker as well as a K+ channel opener. In experiments in which Cl− current activation was specifically brought about by cellular swelling and performed in solutions where Cl− was the major permeant ion, U83757 (300 nm to 300 μm) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K+-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl− currents as well as the activation of a K+-selective current.

Intracellular H+ and Ca2+ modulation of trypsin-modified ATP-sensitive K+ channels in rabbit ventricular myocytes.

ATP-sensitive K+ current (IK.ATP) channels are thought to play a role in the K+ efflux observed in cardiac ischemia. Intracellular acidosis is a prominent early effect in ischemia; therefore, the effects of acidosis on IK.ATP may have certain pathophysiological implications. Increased intracellular proton concentration (pHi) is known to regulate IK.ATP in frog skeletal muscle by increasing open probability. The pHi effect on IK.ATP is not clearly understood in heart because, unlike frog skeletal muscle, low pHi causes IK.ATP run-down in inside-out patches. This would tend to mask any opening effect of low pHi if it exists. Trypsin modification of IK.ATP has recently been shown to prevent run-down in inside-out patches. We used single channel recordings in inside-out patches to study IK.ATP after exposure to trypsin. After trypsin treatment, the open probability of IK.ATP was not sensitive to pHi in the absence of ATP. In the presence of ATP, however, a decrease in pHi consistently increased the open probability of trypsin-modified IK.ATP by reducing ATP inhibition. In the absence of ATP the mean open probability was 0.43 +/- 0.07 at pHi 7.4, and 0.5 mM ATP decreased the mean open probability to 0.03 +/- 0.04 at pHi 7.4, but mean open probability was significantly increased to 0.20 +/- 0.07 at pHi 6.3 (n = 7, p < 0.01). Ca2+ did not affect the activity of trypsin-modified IK.ATP in either the absence or presence of ATP at pHi 7.4. However, Ca2+ was able to antagonize the low pHi effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cardiac sodium channel gating by lysophosphatidylcholine

The effects of lysophosphatidylcholine (LPC) on Na channels have been investigated in inside-out patches of adult rat ventricular cells using the patch-clamp technique. Applying LPC (9-25 microM) to the inner side of the membrane reduced peak Na current (INa) and prolonged the time course of INa inactivation. Both effects increased with duration of LPC exposure. Analysis of single-channel behavior revealed that after 15-20 min of exposure to LPC, Na channels displayed 2 types of gating behavior. One type consisted of normal kinetics and the other consisted of long-lasting burst (LB) of Na channel openings (up to the 300 ms bursts of the test pulse). This modification in gating resulted in the initial appearance of a slowly decaying and later a noninactivating component of INa in the ensemble average current. The slope conductance and reversal potential of these modified channels remained unchanged from control. Overall open time distribution for long bursting kinetics channels was biexponential, indicating existence of two long-lasting bursting modes, one with fast kinetics (LB-f, mean open time 1.2 ms) and one with slow kinetics (LB-s, mean open time 13.7 ms). These data indicate that exposure to LPC results in the slow interconversion of Na channels between several modes of activity, including those in which inactivation occurs slowly or not at all.

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).

Sodium channels in cardiac Purkinje cells

Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3Ω cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes.

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.