Figure 2. Kir2.1 and HCN2 currents counterbalance, generating N-shaped I-V relationships and two levels of resting membrane potential in transfected CHO cells in 2 mM [K⁺]_e.

(A-C) Resting membrane potentials and whole-cell ramp currents of three populations of CHO cells that express both human Kir2.1 and HCN2 channels before and after [K⁺]_e was changed from 5 mM to 2 mM: (A) 35% of the cells remain on hyperpolarization in 2 mM [K⁺]_e, (B) 32% of the cells spontaneously jump into permanent depolarization, and (C) 33% of the cells fluctuated between the two levels.

Top row: Resting membrane potentials. Blue numbers indicate phases 1 and 2 of changes in resting membrane potential. Time scale, 100 s. Insert in B, blockade of HCN2 channels with 30 μM ivabradine eliminated the classical two levels of resting membrane potential; Resting membrane potentials were recorded when bath solutions were first changed from 5 to 2 mM [K⁺]_e, then reversibly applied for 30 μM ivabradine (teal bar), and eventually returned to 5 mM [K⁺]_e.

Second row: (A) Whole-cell ramp currents in 5 (black line) and 2 mM (pink line) [K⁺]_e; (B-C) Pink lines indicate ‘a’ type N-shaped ramp currents with three zero-current potentials in 2 mM [K⁺]_e, blue lines indicate ‘b’ type ramp currents with a reversal potential matching the third zero-current potential of ‘a’ type N-shaped ramp currents, and green line indicates ‘c’ type ramp currents with a reversal potential comparable to the first zero-current potential. Often “a”, “b”, and/or “c” types of currents were recorded in the same cells during recordings of whole-cell ramp currents each 15 s. Dash purple line indicates a linear combination of Kir2.1 and K2P1 whole-cell ramp currents that were individually recorded in 2 mM [K⁺]_e.

Third row: Pink and green lines indicate whole-cell currents before application of channel blockers; purple and orange lines indicate the currents after sequential application of 0.5 mM Ba²⁺ or both 0.5 mM Ba²⁺ and 2 mM Cs⁺ in 2 mM [K⁺]_e, respectively. Ba²⁺ completely blocks Kir2.1 currents, and Cs⁺ inhibits HCN2 currents. The HCN2 currents at −100 mV were −29.2 ± 19.9 pA (n=5) for A, −111.0 ± 42.1 pA (n=3) for B, and −95.1 ± 15.9 pA (n=6) for C.

Bottom row: (A) Resting membrane potentials (filled black and pink bars) of the cells and reversal potentials (open black and pink bars) of whole-cell ramp currents in 5 mM and 2 mM [K⁺]_e; (B-C) Two levels of resting membrane potential (patterned black bars) of the cells, the first and third zero-current potentials (patterned pink bars) of ‘a’ type N-shaped ramp currents, and reversal potentials of ‘b’ (open blue bars) and ‘c’ (open green bar) types of ramp currents in 2 mM [K⁺]_e (n=5–65).