The tarantula toxin GxTx detains K⁺ channel gating charges in their resting conformation

Abstract

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand–protein complexes. Here we investigate how ligand binding to a K+ channel’s voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1’s voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1’s pore opening step does not involve movement of its voltage sensors.

Optical measurement of voltage sensing by endogenous ion channels

“…The fluorescence-voltage response was fit with a Boltzmann function 28 with a half maximal voltage midpoint (V1/2) of -27 mV and a steepness (z) of 1.4 elementary 29 charges (e0) (Fig 4 C, black line). This is strikingly similar to the voltage dependence of 30 integrated gating charge from Kv2.1-CHO cells, without any GxTX present V1/2 = -26 mV, z = 31 1.6 e0 (Tilley et al 2019). The similarity suggests that the degree of GxTX-594 labeling 1 corresponds to the fraction of the channels' voltage sensors that have their gating charges in their 2 most intracellular resting conformation.…”

“…Scheme I 13 assumes that voltage sensor conformational changes equilibrate much faster than ECAP binding 14 or unbinding, such that binding and unbinding are the rate-limiting steps. As ECAP labeling 1 requires seconds to equilibrate (Fig 4 E), and Kv2 channel gating equilibrates in milliseconds 2 (Tilley et al 2019), about 3 orders of magnitude more quickly, the simplifying approximation 3 that voltage sensor conformations are in continuous equilibrium appears reasonable. Kv2 4 channels couple many conformational changes to each other and their gating is more complex 5 than a single voltage sensor conformational change (Scholle et al 2004, Jara-Oseguera et al 6 2011, Tilley et al 2019.…”

“…As ECAP labeling 1 requires seconds to equilibrate (Fig 4 E), and Kv2 channel gating equilibrates in milliseconds 2 (Tilley et al 2019), about 3 orders of magnitude more quickly, the simplifying approximation 3 that voltage sensor conformations are in continuous equilibrium appears reasonable. Kv2 4 channels couple many conformational changes to each other and their gating is more complex 5 than a single voltage sensor conformational change (Scholle et al 2004, Jara-Oseguera et al 6 2011, Tilley et al 2019. However, models which presume independent activation of a voltage 7 sensor in each of the four Kv2.1 subunits accurately predict many aspects of voltage activation 8 and voltage sensor toxin binding (Lee et al 2003, Tilley et al 2019).…”

“…GxTX 10 has a high affinity for resting voltage sensors: 13 nM for rat Kv2.1 in CHO-K1 cells (Tilley et al 11 2014) . When Kv2.1 voltage sensors adopt active conformations, GxTX affinity becomes at least 12 5400-fold weaker (Tilley et al 2019). Fluorescently-tagged derivatives of GxTX label cell 13 surfaces where Kv2.1 proteins are heterologously expressed.…”

“…In response to 1 voltage change, the kinetics of fluorescence change were slower at the center of the basal surface 2 and faster towards the outer periphery ( We suspect that the more 12 extreme location dependence at -80 mV is due to a high density of Kv2.1 binding sites in the 13 restricted extracellular space between the cell membrane and glass coverslip, such that GxTX-14 594 is depleted from solution by binding Kv2.1 before reaching the center of the cell. After 15 unbinding at +40 mV, each GxTX-594 molecule is less likely to rebind to Kv2.1 as it diffuses 16 out from under the cell, because Kv2.1 voltage sensors are in an active, low affinity 17 conformation (Tilley et al 2014(Tilley et al , 2019. This expected difference in GxTX-594 buffering by 18…”