Ancillary subunits and stimulation frequency determine the potency of chromanol 293B block of the KCNQ1 potassium channel

Abstract

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha-subunit of a voltage-gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue-specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage-dependent channel. In epithelia, KCNQ1 co-assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100-fold (IC(50) for KCNQ1 = 65.4 +/- 1.7 microm; KCNQ1/KCNE1 = 15.1 +/- 3.3 microm; KCNQ1/KCNE3 = 0.54 +/- 0.18 microm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency-dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1-like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit-mediated changes in gating can be dissociated from subunit-mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side-effects such as a predisposition to acquired long QT syndrome.

Figure 1. Topological cartoon of the putative structure of KCNQ1 channels and the KCNE subunit

KCNQ1 channels are thought to have six transmembrane segments, with a charged S4 segment responsible for voltage sensing. The region between S5 and S6 forms the H5 loop which plays a role in sensitivity and gating. The KCNE subunits are proteins with a single transmembrane-spanning domain and a cytosolic C-terminal. The details of the interaction between the KCNE ancillary subunits and the KCNQ1 channel are suggested to involve multiple regions of the channel and subunit. The amino acids in the aligned putative transmembrane segments of KCNE1 and KCNE3 are shown. The valine and positionally analogous threonine relevant to the KCNE3[V72T] mutation are indicated in bold.

Figure 2. Representative traces from two-electrode voltage-clamp recordings from heterologously expressed currents in Xenopus oocytes

A standard two-pulse protocol was used. The first pulse (2 s) was from the holding potential of −80 mV to a voltage between −90 and +50 mV in 10 mV steps. The second pulse (1.5 s) was to −60 mV. Traces are from: A, KCNQ1 channels; B, KCNQ1 channels co-expressed with KCNE1; C. KCNQ1 channels co-expressed with KCNE3.

Figure 3. Effect of chromanol 293B on currents

Representative traces showing currents elicited by a 3 s depolarizing pulse from the holding potential of −90 mV to potentials between −80 to +40 mV, followed by a 500 ms pulse to −60 mV, then repolarization to the holding potential. A, KCNQ1. B, KCNQ1 (same oocyte as A) with 50 μm 293B. C, KCNQ1/KCNE1. D, KCNQ1/KCNE1 (same oocyte as C) with 50 μm 293B. E, KCNQ1/KCNE1. F, KCNQ1/KCNE1 (same oocyte as E) with 10 μm 293B. G, KCNQ1/KCNE3. H, KCNQ1/KCNE3 (same oocyte as G) with 1 μm 293B.

Figure 4. Dose–response curve for the effect of 293B on KCNQ1 channels with ancillary subunits

Peak current was measured at the end of a 3 s pulse to +40 mV. Data are fitted by a Hill equation: I = 1 − ([293B]/([293B] + IC₅₀)). The calculated IC₅₀ values are: KCNQ1, 67.0 ± 1.6 μm (▪); KCNQ1/KCNE1, 16.1 ± 1.8 μm (•); KCNQ1/KCNE3, 0.72 ± 0.02 μm (▴). For each point, n = 3 − 8.

Figure 5. Time dependence of 293B block

The fraction of current block during a 3 s depolarization from −90 to +40 mV are shown. Fraction of block is calculated as 1 − I_drug/I_control. Continuous lines are averaged traces (n = 4 or 5) and grey lines represent s.e.m. A, KCNQ1 with 50 μm 293B. B, KCNQ1/KCNE1 with 50 μm 293B. C, KCNQ1/KCNE3 with 1 μm 293B. D, E and F are the same data as shown in A, B and C, but with an expanded timescale and Y axis to enable changes in the first few hundred milliseconds to be seen clearly. G, bar charts to show the difference in percentage block of current at the beginning (filled bar) and end (open bar) of the 3 s pulse for KCNQ1, KCNQ1/KCNE1 and KCNQ1/KCNE3 currents. Only the KCNQ1/KCNE1 current was significantly different at the beginning of the pulse compared with the end of the pulse.

Figure 6. Stimulation frequency dependence of chromanol 293B

A, a 500 ms depolarizing pulse from −90 mV to +50 mV was applied with a 5 s inter-stimulus interval to KCNQ1 channels in the presence of 50 μm 293B and KCNQ1 co-expressed with KCNE1 in the presence of 10 μm 293B. B, a 500 ms depolarizing pulse from −90 mV to 50 mV was applied with a 500 ms inter-stimulus interval to KCNQ1 channels with 10 μm 293B and KCNQ1/KCNE1 with 50 μm 293B. Fractional current is I_{drug(t = x)}/I_{control(t = x)}.

Figure 7. Potentiation of KCNQ1/KCNE1 current with repetitive stimulation

A, peak current elicited by a depolarizing step from −90 mV to +50 mV, normalized to the first pulse following 30 s rest is plotted against time. B, repetitive rapid stimulation of KCNQ1/KCNE1 increases the magnitude of the current in a bi-exponential manner (τ_fast = 1.07 ± 0.04, τ_slow = 7.66 ± 0.43 s). In the presence of 293B the potentiation is a mono-exponential process (τ_293B= 0.87 ± 0.16 s) which is not significantly different from the fast component of the current potentiation in the absence of drug (n = 4, P > 0.2). C, raw traces showing the current from the 1st and 20th sweeps of 1 Hz stimulation. D, ratio of KCNQ1/KCNE1 current with and without 10 μm 293B for the 20th sweep. Also shown are data obtained from similar experiments in the presence of 50 μm 293B. There is relatively little time dependence of drug block during the 20th sweep with either 10 or 50 μm 293B.

Figure 8. KCNQ1/KCNE3[V72T]

A, representative current traces recorded from KCNQ1/KCNE3[V72T] channels using a two-pulse protocol. The first pulse was 3 s depolarization to potentials between −80 and +40 mV from the holding potential of −90 mV. This was followed by a 500 ms step to −60 mV before returning to the holding potential. B, representative trace showing the effect of 1 μm 293B (same oocyte and scale as A). C, the effect of 1 μm 293B on KCNQ1/KCNE1 current is not significantly different from the effect of 1 μm 293B on KCNQ1/KCNE3[V72T] (n = 6, P > 0.6). D, during a 3 s depolarization from −90 mV to +40 mV in the presence of 1 μm 293B, there is a gradual increase in the degree of block KCNQ1/KCNE3[V72T] current. Black trace is average fraction of block from 6 experiments, grey lines indicate s.e.m.

Figure 9. Rate dependence of KCNQ1/KCNE3[V72T]

A, a 500 ms depolarizing pulse from −90 to +50 mV was applied every 500 ms in the presence and absence of 1 μm 293B to test for rate dependence of block. The degree of block did not significantly change during the pulse of 80 rapid depolarizations. B, despite the lack of rate-dependent block, the current is still potentiated by rapid stimulation. C, the increase in current is well fitted with a single exponential curve, regardless of the presence of 1 μm 293B. The two rate constants, τ_Control = 1.11 ± 0.18 s and τ_293B = 1.45 ± 0.72 s, were not significantly different (P > 0.5, n = 3). D, signal averaged ratios showing the development of block during a 3 s depolarizing pulse from −90 to +40 mV when KCNQ1/KCNE1 is exposed to 50 μm 293B and KCNQ1/KCNE3[V72T] is exposed to 1 μm 293B (values chosen to give the similar fraction of block in each channel). The fraction of block is normalized to the total block at the end of a 3 s pulse. The time course of development of the late (> 500 ms) block is similar for KCNQ1/KCNE1 and KCNQ1/KCNE3[V72T].

Figure 10. Models of KCNQ1 kinetic behaviour

A, Seebohm et al. (2003b) proposed this kinetic scheme to describe the complex biphasic gating behaviour in KCNQ1 channels in the absence of ancillary subunits. B, KCNE1 slows activation and increases the sigmoidal delay, suggesting the presence of multiple closed states. With KCNE1 there is no inactivation. Our results suggest that the two open states may have higher affinity than the KCNQ1 open states for chromanol, which gives rise to the time-dependent aspects of block. C, KCNE3 appears to lock the channel in an open state, inhibiting transitions to either the closed or inactivated states. This open state has a very high affinity for chromanol. However, the high affinity site appears unrelated to the portion of the molecule which confers changes of activation gating.