Voltage dependence of ATP-dependent K+ current in rat cardiac myocytes is affected by IK1 and IK(ACh)

Abstract

In this study we have investigated the voltage dependence of ATP-dependent K+ current (I(K(ATP))) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current-voltage relation of 2,4-dinitrophenole (DNP) -induced I(K(ATP)) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and -100 mV. In ventricular myocytes, the background current-voltage relation of which is dominated by a large constitutive inward rectifier (I(K1)), the slope conductance of I(K(ATP)) was reduced at membrane potentials negative to E(K) (around -50 mV), resulting in an outwardly rectifying I-V relation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing to I(K1) channels, in atrial myocytes resulted in a large I(K1)-like background current. The I-V relation of I(K(ATP)) in these cells showed a reduced slope conductance negative to E(K) similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (I(K(ACh))), irreversibly activated by intracellular loading with GTP-gamma-S, the I-V relation of I(K(ATP)) showed a reduced slope negative to E(K), as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge-carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inward I(K(ATP)) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current.

Figure 1. Voltage dependence of I_K(ATP) reconstituted in CHO cells

Cells were cotransfected with vectors encoding for mouse EGFP-Kir6.2-C1 fusion protein and mouse SUR2A. A, current recording from a representative transfection-positive CHO cell showing activation of I_K(ATP) by DNP. B, current recording from a representative cell transfected with the empty GFP vector. The rapid deflections in A and B and in subsequent figures represent changes in membrane current due to voltage ramps from −120 to +60 mV. A scheme of the voltage-ramp protocol applied routinely at 0.1 s⁻¹ to generate I–V relations is illustrated in the inset. C, difference I–V relation obtained by subtraction of the voltage-ramp induced currents labeled a and b in A.

Figure 2. DNP-induced I_K(ATP) in a representative atrial myocyte

A, recording of membrane current. Acetylcholine (ACh) and DNP were applied as indicated (see text for further detail). B, background-subtracted I–V relations of ACh-activated current and DNP-activated current.

Figure 3. DNP-induced I_K(ATP) in a representative ventricular myocyte

A, recording of membrane current. BaCl₂ (2 mm) and DNP were applied as indicated. B, I–V relations of total background current and total current in the presence of DNP. C, difference current (DNP-activated current) from A. D, the traces on the left represent tail currents marked by the arrowheads in A. The right panel shows superimposed tails of the difference current (b − a) and the tail in the absence of I_K(ATP) (a) scaled up to match the peaks.

Figure 4. I–V relations of I_K(ATP) activated by pinacidil or genisteine

A, I–V relations of current activated by 100 μm pinacidil (A) or genisteine (B) in representative ventricular and atrial myocytes. I–V relations were obtained by subtraction of ramp currents in the absence of the activating compounds from ramp currents recorded after activation by either of the compounds. Curves were normalized to match at 0 mV.

Figure 5. Comparison of I_K(ATP) defined as DNP-activated current by background subtraction or sensitivity to glibenclamide (5 μm)

A, slow recording of membrane current. B, current–voltage relations in the absence (a) and in the presence of DNP (b). C, I–V relation of DNP-activated current (○) and glibenclamide-sensitive current (•).

Figure 6. Increase in inward I_K(ATP) by partial block of I_K1

A, recording of holding potential; Ba²⁺ (2 μm) and pinacidil (100 μm) were superfused as indicated. B, superimposed current–voltage relations of pinacidil-activated current obtained by subtraction as in previous figures.

Figure 7. DNP-activated current in atrial myocytes overexpressing Kir2.1

A, current recording from representative mock-infected GFP-positive myocyte. DNP (100 μm) and ACh (20 μm) were applied as indicated. B, current recording from a pAd-Kir2.1-infected myocyte. Ba²⁺ (2 mm) and DNP were applied as indicated. C, I–V relations of background-subtracted I_K(ATP) from A (•) and from B (○). The graphs were scaled to the current level at 0 mV.

Figure 8. Voltage dependence of atrial I_K(ATP) is affected by ‘background’ I_K(ACh)

A, representative recording of membrane current from an atrial myocytes using a pipette filling solution containing GTP-γ-S (500 μm). About 60 s after rupturing the patch the cell was briefly exposed to ACh (20 μm), resulting in irreversible activation of I_K(ACh). I_K(ATP) was activated by superfusion of DNP as indicated B, I–V relations of I_K(ACh) (•) and I_K(ATP) (○) obtained by subtraction of ramp currents as indicated.

Figure 9. Summarized data demonstrating significantly reduced slope conductance of inward I_K(ATP) in ventricular myocytes and atrial myocytes with increased ‘background’ inward rectifying current

Bars represent mean ratios (G_i/G_o) for different groups of cells or treatments (CHO, CHO cells, n = 6; A, atrial myocytes, n = 12; V, ventricular myocytes, n = 12; V Ba²⁺, ventricular myocytes in the presence of 2 μm Ba²⁺, n = 6; A Kir2.1, atrial myocytes treated with pAd-Kir2.1 adenoviral vector, n = 6; GTP-γ-S, atrial myocytes loaded with GTP-γ-S and exposed to ACh, n = 6).