Slow Conformational Changes of the Voltage Sensor during the Mode Shift in Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channels

Abstract

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are activated by hyperpolarizations that cause inward movements of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. If HCN channels are held open for prolonged times (Ͼ50 ms), HCN channels undergo a mode shift, which in sea urchin (spHCN) channels induces a Ͼ50 mV shift in the midpoint of activation. The mechanism underlying the mode shift is unknown. The mode shift could be attributable to conformational changes in the pore domain that stabilize the open state of the channel, which would indirectly shift the voltage dependence of the channel, or attributable to conformational changes in the voltage-sensing domain that stabilize the inward position of S4, thereby directly shifting the voltage dependence of the channel. We used voltage-clamp fluorometry to detect S4 movements and to correlate S4 movements to the different activation steps in spHCN channels. We here show that fluorophores attached to S4 report on fluorescence changes during the mode shift, demonstrating that the mode shift is not simply attributable to a stabilization of the pore domain but that S4 undergoes conformational changes during the mode shift. We propose a model in which the mode shift is attributable to a slow, lateral movement in S4 that is triggered by the initial S4 gating-charge movement and channel opening. The mode shift gives rise to a short-term, activitydependent memory in HCN channels, which has been shown previously to be important for the stable rhythmic firing of pacemaking neurons and could significantly affect synaptic integration.

S4-based voltage sensors have three major conformations

“…The shift of the Q-V curve was found to correlate with the development of slow inactivation of the sodium conductance. Although the same type of shift was later found in L-type Ca channels (10), the Shaker K channel (8), the HERG K channel (11), the bacterial Na channel (NaChBac) (12), and channels in the hyperpolarization-activated cyclic nucleotide-gated (HCN) family (13)(14)(15), it could not, in every case, be associated with entry into the slow-inactivated state of the channel. For example, the inactivation-deficient mutant S631A of HERG showed the same Q-V shift as the wild type (11).…”

Hyperpolarization-Activated Cation Channels: From Genes to Function

“…Recently, it was shown that the voltage-dependent activation of the HCN channel cloned from sea urchin sperm (spHCN) can shift between two modes depending on the previous activity (53,133,246). In mode I, gating charge movement and channel opening occur at very negative potentials, while in mode II, both processes are shifted to Ͼ50 mV more positive potentials (133).…”

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate

“…As an alternative technique to study I Ks voltage sensing, we chose to pursue a fluorescence assay, voltage clamp fluorometry (17), which tracks changes in emission from a fluorophore attached to the extracellular end of the voltage-sensing fourth transmembrane segment of KCNQ1 (S4) as its environment changes during voltage sensing. This method has been used previously to assay voltage sensor movements in other members of the superfamily of voltage-gated cation channels (17)(18)(19).…”