Sesti F and Goldstein SA (1998), Single-channel characteristics of wild-type IKs...

XB-ART-13892

J Gen Physiol 1998 Dec 01;1126:651-63.

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Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome.

Sesti F , Goldstein SA .

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IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel "flicker." At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was approximately 16 pS (corresponding to approximately 0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.

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Species referenced: Xenopus laevis
Genes referenced: arfgap1 kcne1 mink1

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	Figure 2. Single-channel activity recorded from an inside-out patch containing a few wild-type IKs channels in symmetrical 100-mM KCl solution. (A) Current activation at 40 mV from a holding voltage of −80 mV. (B) Current deactivation on return to −80 mV. Expanded traces 1–4 are from the indicated portions of A and B. Data were sampled at 4 kHz and filtered at 1 kHz.
	Figure 3. Activation of wild-type IKs channels in inside-out patches in symmetrical 100-mM KCl solution. (A) Four traces from a patch showing channels opening after depolarization to 40 mV (arrow) from a holding voltage of −80 mV; 600 ms of the 6-s pulse is shown; data sampled at 4 kHz and filtered at 1 kHz. (B) Cumulative current of 30 sweeps of the patch shown in A. (C) Single trace of another patch with many channels by the protocol in B.
	Figure 4. Single-channel activity of IKs channels formed with wild-type or G55C minK in the presence and absence of external Cd2+. Currents were recorded in outside-out patches containing a few channels held at 60 mV in symmetrical 100-mM KCl solution. (A) IKs channels formed with wild-type minK are insensitive to 1 mM Cd2+ (exposure indicated by bar). (B) IKs channels formed with G55C minK are blocked by 1 mM Cd2+ (exposure indicated by bar). (C and D) Expanded traces from the indicated portions of B. Sampled at 4 kHz, filtered at 30 Hz (A and B), or 1 kHz (C and D).
	Figure 5. Gating attributes of KvLQT1 and IKs channels in excised patches. (A) Normalized open probabilities (I/IMax) for KvLQT1 channels (□) and IKs channels (▪). Peak tail current at −60 mV was measured after a 6-s prepulse to voltages of −20 to 80 mV. The curves are normalized to 80-mV prepulse value. Theoretical lines are constructed with the Boltzmann function: 1/{1 + exp[ez(V1/2 − V)/kT]}, where z is the equivalent valence, e the elementary charge, V1/2 the voltage at which the curve is half maximal (see Table I), k the Boltzmann constant, and T the absolute temperature; for KvLQT1 channels, z = 2.0 ± 0.1; for IKs channels, z = 1.0 ± 0.1. (B) Normalized tail currents in a patch at −60 mV. Currents were elicited by a 6-s pulse to 80-mV voltage (inset). Theoretical lines are single exponential fits to the deactivation time course; results from many patches are in Table I.
	Figure 6. Current–variance relationships of IKs channels formed with wild-type and D76N minK subunits. (A) Family of currents recorded in a patch excised from an oocyte coinjected with KvLQT1 cRNA and a 1:1 mixture of wild-type and D76N minK cRNAs. The patch was clamped at −60 mV and depolarized from −20 to 70 mV in 10-mV steps. (B) Family of currents recorded from oocytes coinjected only with KvLQT1 and D76N minK cRNAs. The protocol was similar to A, except that test pulses were up to 80 mV. (C) Current–variance relationships computed from the patches in A (○) and B (•); the conductances calculated in these patches were 8.2 and 4.0 pS, respectively. (D) Current–voltage relationship of peak tail currents measured for D76N IKs channels, mean ± SEM for four patches. (E) Dependence of unitary conductance on the fraction of injected wild-type and D76N cRNAs. The theoretical line was constructed according to Eqs. 4–6 with a best fit yielding a maximal value of n = 2.8 minK subunits per channel (see discussion).
	Figure 7. Current–variance relationships of IKs channels formed with wild-type and S74L minK subunits. (A) Family of currents recorded in a patch excised from an oocyte coinjected with KvLQT1 cRNA and a 1:1 mixture of wild-type and S74L minK cRNAs. The patch was clamped at −60 mV and depolarized from −20 to 100 mV in steps of 10 mV. (B) Family of currents recorded from oocytes coinjected only with KvLQT1 and S74L minK cRNAs. The protocol was as in A with steps from −20 to 90 mV. (C) Current–voltage relationship of peak tail currents measured for S74L IKs channels, mean ± SEM for three patches. (D) Current–variance relationships computed from the patches in A (▿) and B (▾); the conductances calculated in these patches were 14 and 8.7 pS, respectively.
	Figure 8. Current–variance relationships of IKs channels formed with S74L and D76N minK subunits. (A) Family of currents recorded from a patch excised from an oocyte coinjected with KvLQT1 cRNA and a 1:1 mixture of D76N and S74L minK cRNAs. The patch was clamped at −60 mV and depolarized from −20 to 100 mV in 10-mV steps. (B) Current–voltage relationship of peak tail currents measured for channels, mean ± SEM for three separate patches. (C) Current–variance relationship computed from the patch in A; the conductance calculated in this patch was 6.0 pS.
	Figure 9. Gating attributes of mutant IKs channels. (A) Normalized open probabilities (I/IMax) for cells with D76N IKs channels (•, z = 1.3 ± 0.2), S74L IKs channels (▾, z = 1.6 ± 0.1), and S74L-D76N IKs channels (♦, z = 1.3 ± 0.2); protocols, theoretical fits, and equivalent valences (z) as in Fig. 5; the dotted line is the curve for wild-type IKs channels from Fig. 5 A. (B) Normalized tail currents at −60 mV for D76N, S74L, and D76N-S74L IKs channels; the dotted line is the curve for wild-type IKs channels from Fig. 5 B. Currents were elicited after a 6-s test pulse to 80 mV. Theoretical lines were constructed as described in the legend to Table I.
	Figure 10. Currents recorded in CHO cells expressing KvLQT1 or IKs channels by whole-cell configuration. Cells were clamped to −80 mV, and then depolarized for 6 s to test potentials from −60 to 10 mV (KvLQT1) or −60 to 50 mV (IKs) in 10-mV steps. The solutions were (mM): bath: 20 KCl, 100 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES, pH 7.5; pipette: 120 KCl, 2 MgCl2, 2 EGTA, 10 HEPES, pH 7.5. (A) KvLQT1 channels. (B) IKs channels. (C) Variance–current relationships of data in A (□, KvLQT1 channels) and B (▪, IKs channels). The calculated conductances in these patches were 2.2 and 8.2 pS, respectively.
	Figure 11. Calculated unitary conductances as a function of the experimental bandwidth. Curves shown for KvLQT1 channels (□), IKs channels (▪), D76N IKs channels (•), and S74L IKs channels (▾). The curves were obtained measuring the variance at 70 mV, using cut-off frequencies from 0.25 to 25 kHz, and then scaled the value of conductance determined at 25 kHz. Data were sampled at 80 kHz and digitally filtered at the indicated frequencies. Each curve represents the average of three patches.

References [+] :

Ackerman, The long QT syndrome: ion channel diseases of the heart. 1998, Pubmed