Re-tallying transport

Bert de Groot had a problem: too many ions.

As a molecular dynamics specialist at Göttingen’s Max Planck Institute for Biophysical Chemistry, de Groot has been probing the thorny problem of ion channel permeation: how proteins carry specific ions across cell membranes. De Groot joined four other speakers in Saturday’s Permeation & Transport Subgroup session, one of the opening-day events of the 2016 Annual Meeting of the Biophysical Society. Sudha Chakrapani from Case Western Reserve University had kicked off the session with structural and spectroscopic data on pentameric ligand-gated ion channels; de Groot would be followed by talks on transporters by Nieng Yan (Tsinghua University) and Peter Hinterdorfer (Johannes Kepler University), and a broader treatment of membrane transport from the NIH’s Sergey Bezrukov.

It’s been over a decade since Nobel laureate Rod MacKinnon used the simple bacterial protein KcsA to identify ions at four precise positions in the channel pore, specifically coordinated by backbone oxygens in the selectivity filter. And it’s long been assumed that charge repulsion would allow only two of these four positions to be occupied at a time, with alternating spots filled by water molecules. But de Groot’s simulations—using computational electrophysiology, a method to push ions through channels in silico—indicated something different. With both ions and water in the filter, what he saw was “basically a dead channel”: few ions passed through, even in the presence of a strong driving force. A transition occurred when water exited the filter, placing ions in adjacent positions: their repulsion switched the channel into a conducting state, with apparent currents approximating functional recordings. These results were replicated using multiple force fields, water models, and ion parameters—too many ions in the filter, “at odds with what had always been proposed.”

By 2014, de Groot had some experimental evidence to back up his claims. He partnered with Tim Gruene and George Sheldrick at the University of Göttingen, experts in crystallographic structure refinement, to take a second look at previous X-ray data. When refined solely against anomalous diffraction data, the occupancies of four thallium ions—similar in size to potassium, but with a strong anomalous signal—were close to 0.9–1.0 each in the open KcsA filter, consistent with simultaneous occupancy at all four sites.

In newer data presented Saturday, de Groot teamed up with Tom Baukrowitz of the University of Kiel to study a similar phenomenon in K2P channels, another family of potassium-conducting proteins. These channels are voltage-sensitive, despite lacking a canonical voltage-sensing domain. Baukrowitz and colleagues demonstrated that the inactive selectivity filter itself acts as a voltage sensor in K2P channels, carrying a gating charge of 2.2 elementary charge units. Assuming a linear voltage drop, this implies that at positive potentials (where the channel is open), 3–4 ions should be forced into the filter—likely occupying all four binding sites. As described in their paper, published last Thursday in Cell, “this represents the first direct electrophysiological measurement of the number of ions that can simultaneous occupy the filter in a K+ channel.” Through further computational electrophysiology experiments, de Groot demonstrated that mutations reducing ion binding at either end of the filter alter permeation and voltage-sensing: more evidence that four ions may not be too many for normal function, after all.

More than a conference amuse-bouche, subgroups like Permeation & Transport help likeminded researchers focus and mingle before the distraction of posters and overlapping sessions begins the following day. This year, the number of Subgroup Saturday sessions was up to fourteen: newcomers Bioengineering and Cryo-EM held two-hour inaugural symposia, while longstanding participants Mechanobiology and Bioenergetics ran as long as ten hours. And although the ever-expanding Saturday schedule suggests specialization, subgroups generally play well together—e.g. in cosponsoring Sunday evening’s SRAA student poster competition; in a nod to their inherently overlapping nature, each contestant enters in at least two categories. When the transport talks wrapped up for the subgroup business meeting, conference goers moved onto other sessions or into downtown LA with a few more ions in the filter, and a few more ideas for the meeting to come.

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