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Two open states and rate-limiting gating steps revealed by intracellular Na⁺ block of human KCNQ1 and KCNQ1/KCNE1 K⁺ channels

Michael Pusch, Loretta Ferrera and Thomas Friedrich*

1. KCNQ1, the first member of a new K⁺ channel family, associates with the small KCNE1 subunit to form the slow cardiac delayed rectifier current, I_Ks. Mutations in both genes encoding these channels lead to cardiac arrhythmia. We studied the block by intracellular Na⁺ of human homomeric KCNQ1 (homomers) and heteromeric KCNQ1/KCNE1 (heteromers) expressed in CHO cells (Chinese hamster ovary cell line) using whole-cell patch recording.
2. In the nominal absence of extracellular K⁺ and with 65 mM intracellular K⁺, the replacement of 65 mM intracellular N-methyl-D-glucamine (NMDG⁺) by 65 mM Na⁺ induced a decay of outward (K⁺) currents through homomers after maximal activation reminiscent of an inactivation process. The decay had a time constant in the hundreds of milliseconds range.
3. The inactivation process of homomers was, however, not directly dependent on [Na⁺]_i, as evidenced by unaltered biphasic deactivation at negative voltages.
4. An instantaneous voltage-dependent Na⁺ block of homomers was revealed using tail current protocols with activating prepulses that saturated the gating processes of the channel. The instantaneous block was partially relieved at very large positive voltages ( 60 mV) and in 20 mM extracellular K⁺. The instantaneous block of homomers was much less pronounced if the tail currents were measured after short activating prepulses, demonstrating the presence of (at least) two open states: a first, relatively [Na⁺]_i-insensitive and a subsequent [Na⁺]_i-sensitive open state; the current decay reflects the transition between the two open states.
5. Heteromers exhibited a very similar instantaneous block by Na independently of the prepulse duration. Heteromers did not show a Na-induced current decay.
6. Our results demonstrate the presence of two open states of KCNQ1 channels with different [Na⁺]_i sensitivities. The rate-limiting step of homomeric KCNQ1 gating at positive voltages is the transition between these two open states. The rate-limiting step of the gating of KCNQ1/KCNE1 channels appears to be the entry into the first open state.

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