A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification

Abstract

Large-conductance Ca2+-voltage-activated K+ channels (BK channels) control many key physiological processes, such as neurotransmitter release and muscle contraction. A signature feature of BK channels is that they have the largest single channel conductance of all K+ channels. Here we examine the mechanism of this large conductance. Comparison of the sequence of BK channels to lower-conductance K+ channels and to a crystallized bacterial K+ channel (MthK) revealed that BK channels have a ring of eight negatively charged glutamate residues at the entrance to the intracellular vestibule. This ring of charge, which is absent in lower-conductance K+ channels, is shown to double the conductance of BK channels for outward currents by increasing the concentration of K+ in the vestibule through an electrostatic mechanism. Removing the ring of charge converts BK channels to inwardly rectifying channels. Thus, a simple electrostatic mechanism contributes to the large conductance of BK channels.

Fig. 1.

Eight conserved negatively charged glutamate residues ring the entrance to the intracellular vestibule of BK channels, but not the vestibule of low-conductance K⁺ channels. (a) Sequence alignment to GYG of the indicated K⁺ channels for the selectivity filter and inner helix region. Blue indicates residues identical to mouse BK channels (mSlo1), except that E321 and E324 that form the ring of charge are in red. The GenInfo Identifier nos. are: mSlo1, 487797; hSlo1, 26638650; xSlo1, 14582152; dSlo1, 321029; mSlo3, 6680542; cSlo2, 7188777; MthK, 2622639; sKcsA, 2127577; dShaker, 85110; hKv1.1, 1168947; HERG, 7531135. (b and c) Ribbon representation of the crystal structure of the MthK channel obtained with Swiss Protein Viewer from the atomic coordinates by Jiang et al. (31). The residues R93 and E96 in MthK that correspond to E321 and E324 in BK channels are presented as space-filling surfaces. A side view with only two subunits is presented (b), and a view down the pore of the channel from the intracellular side with all four subunits is presented (c).

Fig. 2.

The ring of eight negative charges at the intracellular vestibule doubles the single-channel conductance of BK channels by an electrostatic mechanism. (a) Representative single-channel current records for WT and four different mutant channels at the indicated . The net charge on the ring of charge for WT and mutant channels is indicated. Open (O) and closed (C) current levels are indicated. Membrane voltage is +100 mV; was 150 mM. The open current levels were well defined for all experiments with WT and the various mutant channels with different amounts of net charge, except for the one experimental condition of +8 charges and 150 mM , where the open current level was unstable, perhaps because the channel did not lock into a stable open conformation under these conditions. Increasing the for channels with +8 charges to either 500 or 3.4 M gave stable, well defined open current levels. (b–d) Plots of outward single-channel current amplitudes versus voltage for the WT and mutant channels for the indicated . The net charge in the ring of charge is indicated. The absence of visible error bars indicates the SE is less than the size of the symbol. All plotted points in b–d and the lower dotted line in b indicate current amplitudes estimated by all-point histograms of the single-channel current. The upper dotted line in b indicates the highest current levels that occurred infrequently for +8 charges and 150 mM . These brief higher current levels were measured by hand, as they had little effect on the current histograms. The open circles in b for data from E321D at +30, +50, and +80 mV have been shifted 5 mV to the left so they can be seen. (e) Plots of outward single-channel cord conductance at +100 mV versus net charge in the ring of charge at the indicated . The reversal potentials used to determine cord conductance were calculated from the activities of K⁺ with the Nernst equation and were 0 mV for 150 , -27 mV for 500 mM , and -72 mV for 3.4 M . was 150 mM. For 150 mM the line was only fitted from -8 to 0 net charge, with the remaining point at +8 charge and 150 mM plotting the average of the levels indicated by the dashed lines in b.

Fig. 3.

The ring of eight negative charges has little effect on inward single-channel current amplitudes, and the mutation E321D has no effect on outward single-channel current amplitude. (a) Representative inward single-channel currents from WT and mutant channels. The net charge on the ring of charge for WT and mutant channels is indicated. Membrane voltage was -200 mV. (b) Plots of inward single-channel current amplitude versus voltage. (c) Representative outward single-channel currents for the WT and mutant E321D BK channels. Membrane voltage was +150 mV. Symmetrical 150 mM K⁺ was used in a–c.

Fig. 4.

The ring of charge increases the effective concentration of K⁺ in the intracellular vestibule equivalent to that obtained when the is increased from 150 to 500 mM. (a) Representative single-channel currents from WT and mutant channels at the indicated . The net charge on the ring of charge is indicated. Membrane voltage was +100 mV for records with 150 mM . The voltage was +80 mV for recordings with 500 mM to approximately compensate for the greater driving force with 500 mM . To fully compensate would require a record at +73 mV, but there would be little difference between the records at +73 and +80, as seen in b. The was 150 mM. (b) Plots of single-channel current amplitude versus membrane voltage for the indicated channels. The plotted data points with 500 mM were shifted to the right by 27 mV, the absolute magnitude of the calculated shift in reversal potential, to compensate for the greater driving force on with 500 mM .

Fig. 5.

Removing the ring of eight negative charges converts BK channels to inwardly rectifying channels. Plots of single-channel current amplitudes versus membrane voltage for WT channels and a mutant channel with no charge in the ring of charge. The net charge on the ring of charge for the WT and the mutant channel is indicated by each curve. Symmetrical 150 mM K⁺ was used.