Ion channels and transporters are integral membrane proteins involved in the traffic of ions through cell membranes. On the one hand, channels allow the passage of ions very efficiently, at rates of about tens of millions of ions per second. But those high throughputs occur only when the permeation pathway is accessible for the ions; in other words, when the channel is open. On the other hand, the movement of ions through transporters and pumps is tightly couple to availability (and participation) of other substrates resulting in much slower rates of transport, about 100 to 1000 ions per second. Our main objective is to understand how these proteins function. For example, we would like to understand how ions permeate through these proteins, how these proteins regulate the access to their permeation pathways. ION CHANNELS. We use Voltage-Activated Potassium (Kv) Channels and Cyclic Nucleotide Gated (CNG) Channels as models for our study of ion channels. We express high numbers of these channels in a heterologous system and study the functioning of these proteins by using electrophysiological techniques. With CNG channels, this year we had pursued an interesting relationship between permeation and gating. CNG channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although CNG channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na+ or K+ ions. This property was investigated using rod CNG channels formed by expressing the subunit 1 (or a) in HEK293 cells. In the presence of K+ as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na+. At the single channel level, subsaturating concentrations of cGMP shows that the main effect of the permeant K+ ions is to prolong the time channels remain open without major changes in the closed time distribution. In addition, the maximal open probability was higher when K+ was the permeant ion (0.99 for K+ vs. 0.95 for Na+) due to an increase in the apparent mean open time measured from single channel currents. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K+ ions also prolong the time channels visit the open state. These results are consistent with the idea that permeant ions influence the exit of the channel from the open conformation rather than the cGMP binding steps. Finally, blockers known to interact with the pore of CNG channels respond differently to the presence of Na+ or K+ ions, suggesting that these blockers are able to sense the differences of the pore due to the presence of different ions. Collectively, these results suggest that ions within the pore of CNG channels influence the response to cGMP by changing the stability of the open conformation. In lay terms, this means that while ions are traveling through the ion-conductive pore of the channel, they are interacting with the protein, and these interactions are somehow transmitted to the gating machinery indicating that permeation and gating are intimate processes. With Kv channels and in collaboration with Kenton Swartz (NINDS, MPB Unit), we have successfully measured how many ions permeate through a closed channel. It has long been known that about 1-10 millions ions per second permeate through and open channel. Because the detection limit of standard electrophysiological techniques will allow only modest changes (~100-fold) in ion conduction between open and closed channels to be measured correctly, quantify how many ions permeate in the closed state has been a difficult task. The approach we used was to express the Shaker Kv channel at very high density. Then we measured the macroscopic K+ currents through closed channels at ?100 mV using a pore-blocking toxin that selectively blocks the Kv channels, and in the same cell we counted how many channels were present by integrating the gating currents. We found that a closed channel permeates a mere 100 ions per second. The significance of this result is that the flux of K+ is tightly regulated. The rate at which K+ ions permeate through the channel is decreased at least 100,000 times when the channel is closed. ION PUMPS. We use the Na/K pump as a model for our study on ion pumps. The Na/K pump is a P-type ATPase present in almost all animal cells. In each pump cycle, the protein exports 3 Na+ ions and imports 2 K+ ions, at the expense of one molecule of ATP, and it goes through this cycle at a rate of ~200 times per second. However, the presence of palytoxin somehow alters this cycle and allows the Na/K pump to permeate ions at very high rates, resembling more the behavior of a channel. We would like to understand what are the permeation properties of this channel-like mode of the Na/K pump. For this study, we are using the classical squid giant axon membrane preparation. Preliminary results revealed that the squid Na/K pump responds to palytoxin as Na/K pumps from other tissues do. Addition of palytoxin produces an increase in membrane conductance that is reverted by the presence of ouabain, a highly selective pump inhibitor. Now, the advantage of using the squid preparation is that it allows us to measure the current produced by the palytoxin-modified pump and simultaneously measure the unidirectional flux of Na+ ions. With these two measurements we can determine if ions are permeating independently of each other or not. In addition, we could estimate the minimum number of ions that can be present within the permeation pathway at a particular time. Learning this fundamental permeation property in the palytoxin modified pumps will set the grounds for our future attempt to use this model to investigate where and how ions move through the Na/K pump. Preliminary results obtained this past summer at the Marine Biological Laboratory showed that the flux-ratio exponent, a parameter that indicates the number of ions interacting in the pore is ~1.5, suggesting that more than one ion can occupy the pore at a certain time. Theoretically, changing the ionic conditions of the unidirectional flux should change the value of this coefficient reaching an asymptotic value that reflects the maximal number of ions that can be present simultaneously in the pore. We still do not know this asymptotic value, but the fact that we already know that permeation through this modified pumps is not independent is very encouraging to continue this project during the next squid season.
