Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism

Abstract

The effect of glycosylation on Kv1.l potassium channel function was investigated in mammalian cells stably transfected with Kv1.l or Kv1.1N207Q. Macroscopic current analysis showed that both channels were expressed but Kv1.1N207Q, which was not glycosylated, displayed functional differences compared with wild-type, including slowed activation kinetics, a positively shifted V 1/2, a shallower slope for the conductance versus voltage relationship, slowed C-type inactivation kinetics, and a reduced extent of and recovery from C-type inactivation. Kv1. 1N207Q activation properties were also less sensitive to divalent cations compared with those of Kv1.l. These effects were largely due to the lack of trans-Golgi added sugars, such as galactose and sialic acid, to the N207 carbohydrate tree. No apparent change in ionic current deactivation kinetics was detected inKv1.1N207Q compared with wild-type. Our data, coupled with modelling, suggested that removal of the N207 carbohydrate tree had two major effects. The first effect slowed the concerted channel transition from the last dosed state to the open state without changing the voltage dependence of its kinetics. This effect contributed to the G-V curve depolarization shift and together with the lower sensitivity to divalent cations suggested that the carbohydrate tree and its negatively charged sialic acids affected the negative surface charge density on the channel's extracellular face that was sensed by the activation gating machinery. The second effect reduced a cooperativity factor that slowed the transition from the open state to the dosed state without changing its voltage dependence. This effect accounted for the shallower G-V slope, and contributed to the depolarized G-V shift, and together with the inactivation changes it suggested that the carbohydrate tree also affected channel conformations. Thus N-glycosylation, and particularly terminal sialylation, affected Kv1.l gating properties both by altering the surface potential sensed by the channel's activation gating machinery and by modifying conformational changes regulating cooperative subunit interactions during activation and inactivation. Differences in glycosylation pattern among closely related channels may contribute to their functional differences and affect their physiological roles.

Figure 1. Kv1.1 and Kv1.1N207Q immunoblot analysis

A, immunoblot analysis using Kv1.1 antibodies of membranes isolated from native brain and from Kv1.1 or Kv1.1N207Q stably transfected cell lines. About 20 μg membrane protein from Kv1.1 or Kv1.1N207Q stable transfectants was +/- Endo H or PNGase glycosidase treated and immunoblotted as described in Methods. p80, p60 and p55 represent protein bands of 80, 60 and 55 kDa. In lane 3, cell surface biotinylation/immunoblotting methods were used to determine the glycosylation pattern of Kv1.1 channels on the cell surface as described in the Methods. In lane 11, Kv1.1 Lec1 represents Kv1.1 expressed in Lec1 cells (glycosylation-deficient CHO cells). B, schematic diagram of a Kv1.1 monomer and the amino acid sequence of its extracellular S1-S2 loop.

Figure 2. Kv1.1 and Kv1.1N207Q exhibited differences in activation parameters

A and B, whole-cell current traces for Kv1.1 and Kv1.1N207Q. Eighty millisecond depolarizing pulses were applied from −70 to 50 mV in 10 mV increments. The currents were scaled to equal each other so that activation kinetics can be compared visually. In original traces, Kv1.1 had a peak current of 1500 pA and Kv1.1N207Q had a peak current of 1700 pA at 50 mV. C, normalized G/G_mvs. voltage curves of Kv1.1, Kv1.1N207Q and Kv1.1 in Lec1 cells. Conductance values were plotted as a function of test potential and points were fitted to either a simple (n = 1) or a fourth power (n = 4) Boltzmann equation, G = (G_m/1 + exp[(V_p - V_1/2)/a]ⁿ_, using an automated non-linear least squares curve fitting routine (Levenberg-Marquardt Method). G_m is the maximum sustained conductance, V_p is the membrane potential. D, group data. For a simple Boltzmann equation, V_1/2 was the test potential, where G/G_m= 0.5 and a represented the slope of the voltage dependence of activation (given by a =kT/ze; where T is the absolute temperature, k the Boltzmann constant, e the electron charge, and z the valency of the gating charge). For a Boltzmann equation raised to the 4th power, V_1/2 was the potential at which the channel was half-way through the activation process and the a slope value was the total apparent charge displayed by each subunit during the activation process (Hoshi et al. 1994; Zagotta et al. 1994a,b). **Statistical significance of P < 0.01 in an ANOVA test.

Figure 3. Kv1.1 and Kv1.1N207Q exhibited differences in activation kinetics of ionic currents

A and B, activation rise time as a function of test voltages. Rise time is the required time for each trace to rise from 10 % up to 90 % of its peak current; A is a linear plot and B a log plot. C and D, activation time constants (τ) as a function of voltage. Activating current traces were fitted with a single-exponential function from the time they reached half-maximal amplitude to the maximum current level as a function of test voltages; C is a linear plot and D a log plot. Data were fitted with a decaying exponential function above −10 mV (at potentials where deactivation was minimal); k(0) = activation time constant at 0 mV; a = voltage dependence.

Figure 4. Kv1.1 and Kv1.1N207Q exhibited no differences in deactivation kinetics of ionic currents

A and B, averaged normalized deactivation currents are shown for both Kv1.1 and Kv1.1N207Q. Cells were held at −80 mV and depolarized to 20 mV for 80 ms followed by repolarizations to different test potentials indicated beside the traces (mV). Data were normalized to the value at 1 ms to avoid contributions from residual capacitance transients. C and D, deactivation time constants (τ) as a function of test voltage. Individual deactivation current traces were fitted with a single exponential and the time constants were plotted as a function of voltage in C as a linear plot and in D as a log plot. Data were fitted with a decaying exponential function below −50 mV (where activation was minimal). k(0) = deactivation time constant at 0 mV; a = voltage dependence.

Figure 6. Kv1.1 and Kv1.1N207Q exhibited differences in recovery from C-type inactivation

A and B, superimposed whole-cell current traces elicited by a series of double-pulse protocols. The first depolarizing pulse was to 20 mV for 10 s, then an 80 ms pulse to 20 mV with holding intervals from 100 ms to 30 s at −80 mV (sampling rate 5–10 kHz and filtered at 1–2 kHz depending on the protocol's length). C, because of the different magnitude of C-type inactivation, recovery processes were evaluated using a normalized ratio (A, inactivated current in the first 10 s pulse; B, recovered current during the various interval period). D, normalized recovery ratio as a function of the intervals of the double-pulse protocols, showing a slower recovery process of Kv1.1N207Q compared with wild-type.

Figure 5. Kv1.1 and Kv1.1N207Q exhibited differences in C-type inactivation parameters

A and B, whole-cell recordings showing C-type inactivation at 20 mV. A double exponential fitted the current decay very well. Inactivation was examined further with long pulse protocols: depolarizing to −20, 0, 20 and 40 mV from −80 mV for 10 s with a sampling rate of 10 kHz (filtered at 2 kHz). C, inactivation ratio is defined as shown. D, inactivated ratio of Kv1.1 and Kv1.1N207Q at 10 and 3 s at each test voltage (*P < 0.05, **P < 0.01 vs. Kv1.1, Student's unpaired t test). Within the same time group containing four different voltages no significant difference was observed. E, inactivated ratio as a function of time (at 20 mV). At every time point significant differences were observed (P < 0.01) between Kv1.1N207Q and Kv1.1. F, inactivating traces at 20 mV for 10 s (A and B) were fitted with a double-exponential function. A significant difference was apparent in the slow component (τ_slow) and its magnitude.

Figure 7. Extracellular strontium affected the V_1/2 for Kv1.1 more than that for Kv1.1N207Q and the former had a higher effective surface charge density than the latter

A and B, steady-state activation versus voltage curves for Kv1.1 and Kv1.1N207Q with and without external Sr²⁺ (20 mM, n = 7; 50 mM, n = 10). C, table of group data for V_1/2 mean values ±s.e.m. V_1/2 values were used to calculate the effective surface charge densities associated with Kv1.1 and Kv1.1N207Q, as described in the Methods. ΔV_1/2, V_1/2 shifts (in mV).

Figure 9. Activation ionic current traces and simulations for Kv1.1 and Kv1.1N207Q

A-D, activation ionic current traces at different voltages (mV) of Kv1.1 and Kv1.1N207Q and simulations using the three models described in the text.

Figure 8. Data and simulations of steady-state activation versus voltage curves of Kv1.1 and Kv1.1N207Q

A-C, steady-state activation versus voltage curves for Kv1.1 and Kv1.1N207Q and simulations from models developed for Shaker-type K⁺ channels (Hoshi et al. 1994; Zagotta et al. 1994a,; see text and appendices for description of these models).

Figure 10. Deactivation ionic current traces and simulations for Kv1.1 and Kv1.1N207Q

Deactivation ionic current traces at different voltages of Kv1.1 (A) and Kv1.1N207Q (B), and their voltage dependence, were fitted by simulation using the ZHA Q-elec model described in the text.