Voltage-dependent block by internal spermine of the murine inwardly rectifying K+ channel, Kir2.1, with asymmetrical K+ concentrations

Abstract

Effects of internal spermine on outward single-channel currents through a strongly inwardly rectifying K(+) channel (Kir2.1) were studied at asymmetrical K(+) concentrations (30 mm external and 150 mm internal K(+)). The current-voltage (I-V) relation for the single channel was almost linear and reversed at -37 ± 3 mV (V(R); n = 19). The channel conductance was 26.3 ± 1.3 pS (n = 24). The open-time and closed-time histograms were fitted with a single exponential function. Internal spermine at a concentration of 1-100 nm reduced the open time of the outward currents in a concentration-dependent manner and produced a blocked state. The steady-state open probability of the outward current decreased with larger depolarizations in both the absence and presence of internal spermine. The steady-state open probability with asymmetrical K(+) and symmetrical (150 mm external and internal K(+)) concentrations plotted against driving force (V - V(R)) coincided with smaller depolarizations in the absence of spermine and larger depolarizations and higher spermine concentrations in the presence of spermine. The blocking rate constants and unblock rates with 30 mm and 150 mm external K(+) were similar at the same driving force. The dissociation constant-membrane potential relation for 30 mm external K(+) was shifted in the negative direction from that for 150 mm external K(+) by 36 mV. These results suggested that the blocking kinetics depends on driving force to produce driving force-dependent inward rectification when the equilibrium potential for K(+) is altered by changing external K(+) and that the energy barriers and wells for blocking ions from passing or lodging are not stable but affected by external K(+) ions.

Figure 1. Single-channel currents and I–V relation in the absence of internal blockers with 30 mm external and 150 mm internal K⁺ concentrations

A, currents were filtered at 0.2 kHz and sampled at 1 kHz. The dotted line indicates the zero-current level. The numbers to the left of each trace refer to the holding potential. B, I–V relationship obtained from the same patch as in A. The slope conductance was 26.6 pS.

Figure 2. Kinetic analysis in the control

Open-time and closed-time histograms constructed for currents at −10 mV. Both are fitted with a single exponential function with the time constant indicated. The last bin of the closed time histogram includes events longer than 100 ms.

Figure 3. Effects of internal spermine on outward currents with a reduced external K⁺ concentration

A, outward currents recorded at −15 mV in the absence and presence of spermine. Spermine decreased the open time dependent on the concentration and induced a blocked state of 250–600 ms duration. B, currents in the presence of 100 nm spermine. The numbers to the left of each trace refer to the holding potential. Inward currents were not affected.

Figure 4. Steady-state open probability–membrane potential relations

Upper panel, smooth lines are fits of a Boltzmann function to average data (n= 3–9). The voltages of half-activation and slope factors were respectively +4.7 mV and 9.9 mV in the control (▪), −13.3 mV and 4.1 mV with 1 nm spermine (•), −21.3 mV and 3.0 mV with 10 nm spermine (▴), and −29.2 mV and 3.2 mV with 100 nm spermine (▾). Middle and lower panels, the steady-state open probability was plotted against driving force. For simplicity, s.d. was omitted. •, 30 mm external K⁺; ▪, 150 mm external K⁺. The data with 150 mm external K⁺ are the same as in Fig. 2B of Matsuda et al. (2003).

Figure 5. Analysis of blocking kinetics

Histograms of open and zero-current times constructed for outward currents in the presence of 10 nm spermine at −15 mV (left) and 100 nm spermine at −15 mV (middle) and −10 mV (right). The open-time histogram was fitted with a single exponential function. The mean open time decreased in a concentration-dependent manner. The currents were filtered with 0.2 kHz and sampled at 1 kHz. Spermine induced a component of zero-current times with the time constant indicated, which represents blocked times. Depolarization at 5 mV prolonged the mean blocked time threefold.

Figure 6. Dissociation constants in the spermine block

A, the open probability was normalized to that in the absence of spermine and plotted against the spermine concentration on a logarithmic scale. The curve was fitted by saturation kinetics with a Hill coefficient of 1 and a dissociation constant of 7.1 nm at −20 mV (•) and 0.53 nm at −10 mV (▪). B, dependence of the dissociation constant on membrane potential. In a semilogarithmic plot, data at 30 mm external K⁺ (•) were fitted by a straight line almost in parallel with that fitted to data at 150 mm external K⁺ (▪) 36 mV apart.