Inactivation mechanisms in voltage-gated ion channels

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Ion channels are water-filled, protein-lined pores that can provide a pathway for inorganic ions across the cell membrane, a process that is fundamental to electrical signaling in muscle and nerve cells. In voltage-gated ion channels, changes of the membrane voltage trigger a process known as gating, which involves conformational changes in the pore that either permit or prevent conduction. Activation gating refers to the opening, in response to membrane depolarization, of a gate in the inner mouth of the channel pore that permits ions to move through the pore at rates as high as millions of ions per second. Typically, however, this conducting state of the channel is self-limited by virtue of a second pore gate, referred to as an inactivation gate, whose closing stops conduction. Inactivation therefore plays an important part in the regulation of the amplitude and duration of current during a depolarizing response. The focus of this proposal is on a voltage-gated channel that allows potassium ions (K+) to pass through it. It is known as the KV4 channel, and it plays a vital role in determining the rhythm and strength of human heart muscle contractions and in modulating chemical communication between neurons in the brain. These functional roles reflect a key property of the KV4 channel: after the activation gate opens the pore conducts, on average, for only a few tens of milliseconds before it becomes non-conductive due to inactivation. In the large majority of voltage-gated ion channels the closing of the inactivation gate is, after a variable delay, coupled to channel opening, i.e., opening of the activation gate triggers a conformation change that enables the inactivation gate to close. But that is not the case in KV4, where the predominant inactivation process occurs when the channel is closed. The hypothesis to be tested is that the KV4 channel has no inactivation gate per se and that inactivation instead represents a disconnection or slippage between membrane depolarization and activation gate opening. Experimental details are provided for two projects. Project 1 pursues the long-known but heretofore overlooked observation that in KV4 the drug 4-aminopyridine (4-AP) prevents inactivation. This is a potentially significant clue because in other KV channel types 4-AP acts, via a binding site in the central cavity of the channel pore, to stabilize the activation gate in its closed conformation. The main question to be addressed by using point mutations in the pore is whether 4-AP binding in KV4 channels also occurs in the central cavity of the pore. Experimentally-derived data of the effects of 4-AP in wild type and mutated KV4 channels will then be incorporated in a numerical simulation of KV4 gating as a test of the hypothesis. Project 2 exploits the results of structure-function studies in Shaker, the prototypical KV channel, which have identified interfaces with the pore and sites within the pore whose mutation stabilizes the activation gate in its closed state. As for the studies with 4-AP, the rationale for the experiments is that mutations that stabilize the closed conformation of the activation gate will inhibit or prevent the transition from the closed to the closed-inactivated state. The goal is to analyze a number of KV4 channel mutations to see if inactivation is compromised. This research is expected to provide important insights into KV4 channel biophysics, and in particular whether activation gate slippage is a viable explanation of its inactivation behaviour. Whether the activation gate slippage hypothesis is supported or rejected, the research is expected to be of wide interest to ion channel biophysicists since closed-state inactivation is known to occur in voltage-gated sodium and calcium channels.