The gating mechanisms of cation channels. Ion channels are integral membrane proteins that allow ions to cross the membrane at rates near the diffusion limit. These channels come in a variety of forms, each carefully adjusted through evolution to perform a particular function in a cell. We focus our attention on the gating properties of two types of ion channels: voltage-activated potassium channels (KV) and cyclic nucleotide-gated channels (CNG). KV channels open and close a K-selective pore in response to changes in the membrane potential. Consequently, they are key players in the generation and propagation of action potentials, as well as in defining the firing pattern of a neuron. These channels are tetramers formed by the co-assembly of four homologous subunits, each containing six predicted transmembrane segments (S1-S6). From a large set of functional and structural studies, two protein domains have been defined: an ion-conducting pore and a voltage-sensing domain. The last two transmembrane segments (S5-S6) and the loop that connects them (known as the P-region) form the actual ion-conducting pore domain. Amino acids of the selectivity filter located in the P-region together with amino acids of the intracellular part of S6 constitute the actual permeation pathway. Segments S1-S4 form the voltage-sensing domain. The main component of the voltage sensor is believed to be S4, a segment rich in positively charged amino acids. In response to a membrane depolarization, a conformational change within the voltage sensor triggers opening of the ion conduction pore to allow K ions to flow passively down their electrochemical gradient. When the membrane is held at negative potentials, ions encounter a large energetic barrier for permeation, a state referred as closed. This process of opening and closing the channel is commonly known as activation gating. The activation gate has been located at the intracellular end of the S6 segment. How tight is this closure point? In other words, are ions allowed to permeate at all when the channel is closed? We have used a combination of macroscopic ionic current, toxin blockade and gating currents to define, more precisely, the conductance of the closed state in wild-type channels. We found that closed channels conduct ions at rates ~100,000 smaller than open channels Despite their overall similarity to KV channels, CNG channels exhibit very different functional behavior and they serve unique roles in the transduction of visual and olfactory information. Their function is to sense variations in the intracellular concentration of cyclic nucleotide that occur in response to either visual or olfactory stimuli. In many ways, CNG channels are similar to KV channels. They are tetramers with each subunit containing 6 transmembrane segments and a P-region between S5 and S6. The main differences are that CNG channels poorly select among cations and the channels are not gated by voltage. Instead, they open and close the pore in response to changes of the intracellular concentrations of cGMP or cAMP. Sensitivity to cyclic nucleotides is conferred by the presence of a cyclic nucleotide binding domain at the C-terminus of each subunit. How the CNG channels open and close their pore is poorly understood compared to the KV channels. The permeation properties of CNG channels have been explored in detail, yet the interactions between permeation and gating, which are well established in KV channels, have been largely ignored. I performed a thorough study on the effects of permeant ions on gating by cGMP and I found that there is an intimate relationship between permeant ions and the gate that opens and closes the channel. Where is this gate in CNG channels? Some evidence suggests that the activation gate in CNG channels might not be in the intracellular side of the S6 transmembrane segment, as we found in KV channels. For example, tetracaine appears to block the permeation pathway of the channel, however, this blocker prefers the closed channel, in sharp contrast to blocker studies in KV channels. In addition, experiments examining the state-dependence of cysteine modification by MTS reagents or Ag ions failed to show dramatic differences between open and closed states in the inner vestibule region (intracellular side of S6), in contrast to what we had observed in KV channels. What structural differences between KV and CNG channels might account for these disparate observations? Could it be that the closed state of CNG channels has a higher conductance than we measured for KV channels? Might the gates in these two related cation channels be located in very different regions? We are now further exploring these questions by combining the use of blockers, cysteine substitution, chemical modification and the ability of the blocker to protect modification. The use of quaternary ammonium (QA) derivatives as a tool to understand protein function has been successful used in the K channel field. These studies revealed that the gate is located at the intracellular side of the channel and that a cavity large enough to accommodate a molecule of ~ 8 A? in diameter is positioned above the gate. With the hope that the use of QA compounds could provide us with useful information on the functioning of CNG channels, we have embarked in a detailed study of the interactions between these compounds and CNG channels. We are presently characterizing the blockade of CNG channels by QA compounds, to proceed later with protection experiments. We expect that our results will provide solid evidence of the role of S6 in opening and closing the channel. Regulation of channels and transporters by RNA editing. The enzymatic conversion of adenosine to inosine in precursor mRNAs allows multiple protein products from a single gene. This increase in genomic capability does not appear to be random, rather it is targeted to functional relevant regions of a protein. The classical example is in the GluRB subunit of the glutamate-gated ion channel, important for fast excitatory synaptic transmission in the central nervous system. Editing underlies the conversion of Q to R in the channel?s pore, rendering the receptor impermeable to calcium ions. Although editing seems to be common among membrane proteins, the functional consequences of editing had been explored in only a few examples. We have characterized the substrate requirements and the functional consequences of an I to V conversion within the intracellular cavity of the human KV1.1 channel. The functional consequence of editing was targeted to fast inactivation, an additional gating mechanism of KV1 channels. This study provides us a guide to ask previously inaccessible questions about the interactions between the inactivation particle and the ion conduction pore. Transporters are essential for ion channel function because they provide and maintain the ionic gradient that allows ions to diffuse through channels. Interestingly, there is no description in the literature of RNA editing in transporters. We are taking advantage of the apparent high levels of editing in squid to examine RNA editing in transporters, initially focusing our attention on the Na/K pump. At present, we have cloned the full cDNA and genomic versions of the Na/K pump from the squid Loligo opalescens, and we have found at least four potential RNA editing sites. We are attempting heterologous expression of the squid pump to enable functional characterization of these sites in order to develop a mechanistic understanding of how editing alters protein function. In addition, we expect that this study will lead us to important structure-function aspects of the Na/K pump, a protein for which our understanding of the structure and function lags far behind most ion channels.

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Wild, Philipp S; Felix, Janine F; Schillert, Arne et al. (2017) Large-scale genome-wide analysis identifies genetic variants associated with cardiac structure and function. J Clin Invest 127:1798-1812
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