Single channel analysis reveals different modes of Kv1.5 gating behavior regulated by changes of external pH

Abstract

In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk-) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single channel current amplitude at 100 mV (1.7 +/- 0.2 pA at pH 7.4, 1.7 +/- 0.2 pA at pH 6.4) and the single channel conductance between 0 and 100 mV (11.8 +/- 0.6 pS at pH 7.4 and 11.3 +/- 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s(-1), to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of channel availability and inactivation.

FIGURE 1

The single channel current amplitude of Kv1.5 does not change between pH 7.4, 6.4, and 5.9. (A) Representative unitary current through a one-channel, outside-out patch at pH 7.4. Current traces were evoked by 1-s depolarizing pulses at 100 mV from a holding potential of −80 mV every 15 s. Kv1.5 unitary currents show flickering behavior. The failure of the channel to open (e.g., last trace in pH 7.4 column) was an infrequent observation at this pH. The corresponding cumulative all-points histogram based on traces in panel A is shown at the bottom of the panel. Fitting the histogram to a three-component Gaussian function gave a value of 1.7 pA for the major conducting level. (B) Unitary current from the same outside-out patch using the same voltage protocol after switching to external solution at pH 6.4. The open channel current does not change but there are more null sweeps. The all-points amplitude histogram shown at the bottom gave a single channel current of 1.7 pA for the major conducting level. The higher proportion of the nonconducting points reflects the higher proportion of null sweeps and the more frequent termination of active sweeps by a long-lived nonconducting state. (C) Unitary current from a different outside-out patch using the same voltage protocol as in panel A but with an external solution at pH 5.9. Channel activity is observed in only one of the 12 sweeps. This dramatic decrease of channel activity was not due to the loss of the channel from the patch since recovery of activity was obtained after returning to pH 7.4 solution (D). The all-points histograms at the bottom of panels C and D give a value of 2.0 pA for the mean open channel current.

FIGURE 2

Open channel current-voltage relationship showing the single channel conductance does not change with pH. (A) Representative unitary currents at voltages between 0 and 100 mV in 20 mV increments at pH 7.4 and 6.4. All traces shown were digitally filtered at 1 kHz. (B) Fitting a line to the i-V relationship at pH 7.4 (○) and pH 6.4 (•) gave a slope conductance (mean ± SD) of 11.8 ± 0.6 pS and 11.3 ± 0.8 pS, respectively. These values for the slope conductance were not significantly different.

FIGURE 3

Current behavior at the macroscopic level and the ensemble average of unitary current of Kv1.5 at pH 7.4, 6.4, and 5.9 are qualitatively similar. (A) Representative, superimposed macroscopic currents at pH 7.4 (top trace), pH 6.4 (middle trace), and pH 5.9 (bottom trace) evoked with a 1 s depolarizing pulse to 100 mV from a holding potential of –80 mV. Peak current was reduced by ∼22% at pH 6.4 and 81% at pH 5.9. Fitting the currents at pH 7.4, 6.4, and 5.9 to a single exponential function gave mean inactivation time constants of 558 ± 75 ms, 556 ± 61 ms, and 346 ± 46 ms, respectively. Normalized currents are shown in the inset to better illustrate the acceleration of inactivation. (B) Ensemble open probability generated from idealized single channel records at pH 7.4, 6.4, and 5.9. The ensemble behavior reproduces the changes of the peak amplitude and kinetics observed in the macroscopic currents. Compared to that at pH 7.4, the peak ensemble current was reduced by 34% at pH 6.4 and 82% at pH 5.9. A single exponential fitted to the ensembles gave time constants of 548, 401, and 197 ms for pH 7.4, 6.4, and 5.9, respectively. Normalized ensembles are shown in the inset. (C) A reduction in channel availability accounts for the reduction of peak macroscopic current by external H⁺. Channel availability (•) agrees well with the normalized relative g_max recorded in HEK293 cells (▵) or the normalized peak macroscopic current in ltk⁻ (○). Availability is defined as the proportion of sweeps with one or more open events. Data from HEK293 cells were obtained from our previous study (3). Briefly, whole-cell currents were recorded from a series of 300-ms depolarizing steps at −50 to +60 mV, and the instantaneous tail currents at −50 mV were analyzed to give the relative g_max values at different pHs. The composition of the bath and pipette solutions were identical to that listed in Materials and Methods except the bath solution contained 5 mM K⁺ and 138.5 Na⁺. Fitting the whole-cell data to the Hill equation gave a pK_H of 6.2 ± 0.2 with a Hill coefficient of 1.6 ± 0.4 (dashed line) for the reduction in relative g_max. Fitting channel availability to the Hill equation (solid line) gave a pK_H of 6.4 ± 0.2 with a Hill coefficient of 1.9 ± 0.2.

FIGURE 4

Modal gating of Kv1.5 at different pHs. (A) A diary plot of Kv1.5 constructed by plotting the open probability per sweep for 150 consecutive sweeps depolarized at 100 mV for 150 ms every 3 s at pH 7.4 with a cell-attached patch. In this example, only one sweep (No. 79) shows mode U gating. (B) Diary plot from another cell-attached patch using an identical protocol but at pH 6.4. Mode A (available) and mode U (unavailable) gating appear in clusters, as suggested by runs analyses (see text). (C) Frequency histogram of the number of consecutive sweeps showing mode U gating at pH 6.4. The length of runs with mode U gating was pooled from seven patches, and the resulting histogram was fitted to a single exponential distribution to give a time constant of 2.8 sweeps (8.4 s). (D) Frequency histogram of the number of consecutive sweeps with mode A gating at pH 6.4. Fitting the histogram to a single exponential distribution gave a time constant of 1.9 sweeps (5.7 s).

FIGURE 5

Decreasing external pH to 6.4 decreases the mean burst length and increases the apparent interburst duration. (A) Representative unitary currents at pH 7.4 in a cell-attached patch. High bath solution was assumed to depolarize the cell to 0 mV; the pipette solution contained 3.5 mM K⁺. Contiguous traces of the first 80 s of activity at 100 mV are shown. Bursts of flickering channel behavior were bracketed by gaps longer than 20 ms (see Materials and Methods). (B) Representative contiguous traces from another cell-attached patch but at pH 6.4. The within-burst behavior was only slightly changed, the mean burst duration was decreased, and the apparent interburst duration was increased. Both traces were digitally filtered at 1 kHz.

FIGURE 6

Extracellular acidification does not significantly affect gating transitions within bursts. Unitary current from cell-attached patches recorded at pH 7.4 and 6.4 with 2- or 3-min depolarizing pulses to 100 mV were idealized using a half-amplitude method. (A) The open duration histogram obtained from the current trace shown in Fig. 5 A was fitted to a biexponential distribution to give time constants of 0.5 and 2.6 ms, and an area of 0.30 and 0.70, respectively. (B) The closed duration histogram obtained at pH 7.4 was fitted to a four-component exponential distribution, and the fastest three time constants were 0.2, 0.5, and 4.8 ms, with a respective area of 0.63, 0.36, and 0.02. The slowest component (time constant 0.48 s) is thought to represent one or more inactivated states. It has a relative area <1% of the total closed events. (C) The open duration histogram generated from the current trace shown in Fig. 5 B at pH 6.4. The fast and slow time constants are 0.5 and 2.1 ms with an area of 0.2 and 0.8, respectively. (D) The closed duration histogram at pH 6.4 was fitted to a four-component exponential distribution. The three fastest components have time constants 0.2, 0.4, and 2.6 ms and an area 0.81, 0.18, and 0.01, respectively. The slowest component (time constant 5.5 s) represents <1% of the total nonconducting events.