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The Na+/K+-ATPase is a chemical molecular machine responsible for the movement of Na+ and K+ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na+/K+-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na+ out of the cell, then imports 2K+ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell's energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades' worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na+ and K+ ions through the Na+/K+-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.

Several mutations in Cx26 promote keratitis ichthyosis deafness (KID), a rare disease in which patients exhibit deafness, skin and corneal disorders. Some KID mutations are located in the amino terminal segment of Cx26, which has been proposed as the putative voltage sensor. Most syndromic mutations elicit a gain of hemichannel function, however the mechanism underlying this phenomenon remains unclear. Here we examined the biophysical properties of the mutant G12R at macroscopic and single channel levels. We perform two-electrode voltage clamp to study the deactivation kinetics and apparent affinity for calcium of hemichannel currents. In contrast to wild-type (wt) Cx26, we found that the activation kinetics of the mutant lack saturation of the current upon depolarization pulses above +40 mV. Estimates of the deactivation time constant yield values of 5s and 10s for G12R and wtCx26 hemichannels, respectively; however the apparent affinity for calcium remains similar to wtCx26. Single channel recordings analysis showed that the mutant virtually eliminates the transitions to the subconductance state. Moreover, although the single channel conductance was similar to wtCx26 hemichannels (320 pS), the open probability and mean open times of G12R hemichannels become larger. Molecular dynamics indicates that the G12R induce an electrostatic effect and ddG calculations support protein destabilization.Supported by Fondecyt-3150634 to IEG; Fondecyt-3140590 to GC; Fondecyt-1130855 to ADM; Anillo-ACT1104 to ADM and CG; Fondecyt-1120802 to CG; Grant RO1-GM099490 to J.E.C; AP has a Conicyt Fellowship. The Centro Interdisciplinario de Neurociencias de Valparaiso is a Chilean Millennium Institute (P09-022-F).

Large-conductance Ca2+ activated potassium channels (BK), which are synergistically modulated by voltage and intracellular calcium, play important role in the control of the cell excitability. Here we report data from a de novo missense mutation in KCNMA1, encoding the BK channel α subunit found in a patient with progressive cerebellar ataxia. The mutation changes the residue of glycine 354, in the selectivity filter signature sequence 352-TVGYG-356 in potassium channels, into a serine. We expressed BK and G354S mutant BK (BK-G354S) channels in Xenopus oocytes and we analyzed macroscopic and on-cell single channel potassium currents recorded under voltage clamp. The macroscopic potassium currents are reduced in magnitude by about ten times and are slowly activated in oocytes expressing BK-G354S channels when compared with oocytes injected with cRNA for BK channels. The microscopic single channel conductance in BK-G354S is also depressed to about a tenth of the single channel conductance of BK channels, which suggest that the smaller macroscopic currents recorded with BK-G354S channels is not a mere problem of expression in the membrane. Actually, as revealed by expressing both proteins in mammalian cells and measuring their expression level using immunofluorescence techniques, BK-G354S channels are expressed in comparable levels with the expression of the wild-type BK channels. The mutation G354S in BK channel significantly decreases its potassium conductance, which importantly denotes a phenotype with loss of function. Biophysical data presented here provides crucial information that helps understanding the neurologic pathophysiology in the affected patient and it may also help in designing possible treatments for this and other similar cases. Supported by NIH GM030376, NS094665-01 and NS082788-02.

The voltage-gated proton channel (Hv1) is a dimer of subunits containing four transmembrane domains (S1-S4) which play the double role of harboring the voltage-sensing charges and forming the proton conduction pathway. Some biophysical properties of Hv1

The most well-known role of Connexins (Cxs) is to communicate the cytoplasm of two adjacent cells forming a gap junction channel in the apposition zone. On the other hand, Cxs form the so called “hemichannels”, a fully functional voltage-gated non-selective ion channel, which present two gating mechanism called “slow” and “fast” gating. Hemichannels consist of 6 monomers, each one composed of four transmembrane domains. Despite the fact that hemichannels are voltage-activated, they lack the classical voltage-sensing domain described for canonical voltage-gated channels. Furthermore, the molecular determinants associated to the voltage activation in connexins hemichannels are still elusive. In this work we search for the molecular determinants associated to voltage detection in human Cx26 hemichannels. Using two electrode voltage-clamp in Xenopus laevis oocytes, we study how the steady-state conductance-voltage (G/V) relationship and kinetics of gating are affected by the neutralization of different charged residues located within the first transmembrane domain. We have found that Cx26 conductance-voltage curve shows an apparent number of gating charges (zδ) of 1.4 e0 for the slow gating mechanism. On the other hand, we obtained a zδ of 3.9 e0 for the fast gate, which is in agreement with the zδ of Cx26 gap junctions. A simple three-state closed-open1-open2 kinetic model is able to account for the channel voltage dependence. Furthermore, our data shows that neutralizing the K41 residue (K41N) increases the zδ; of the slow gate to 2.5 e0, and decreases the zδ of the fast gate to 2.3 e0.This work is supported by FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.), 1130855 (A.M.); ANILLO Grant ACT1104 (C.G.); Beca para Estudios de Magister en Chile Ano 2014 (B.P.); CINV is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turismo.

Regulatory β and γ subunits are responsible for conferring functional diversity to BK channels but little is known about the detailed way that accessory subunits modulate the structure of the pore forming α subunit. It is known that the γ1 subunit produces a large leftward shift of the open probability vs. voltage curve in the absence of internal Ca2+ (Yan and Aldrich, 2010). To explore the external architecture of α subunit in the presence of γ1 subunit, we used lanthanide-based resonance energy transfer (LRET) as a molecular ruler to measure intra- and inter-molecular distances. We introduced a genetically encoded lanthanide binding tag (LBT) that binds Tb3+ (LRET donor) with high affinity at different positions in the α subunit (N-terminal, S0, S1 and S2) and γ1 subunit. Fluorescent probe BODIPY linked to a scorpion toxin was used as LRET acceptor. LRET sensitized emission (SE) decays were analysed using a nano-positioning system that determines the position of LBT-tagged sites with respect to the fixed acceptor near the pore axis. Interestingly, the external architecture of the BK α subunit is modified when co-expressed with the regulatory γ1 subunit indicating a conformational change of the BK voltage sensor domain of the BK channel. The largest changes was in S1 position (∼25 Angstroms) followed by S0. In addition, all γ1-LBT positions were found peripherally positioned with respect to the α subunit.Fondecyt Grant 1110430 (To R. L.)

BK channels are ubiquitously expressed and are involved in the regulation of vascular smooth muscle contractility. Changes in the expression of the BK channel or its auxiliary β subunits are associated with several physiological abnormalities including hypertension. It is known that 17β-Estradiol (E2) activates the BK channel, causing a membrane hyperpolarization and in vascular tissue, through its action on BK channels, decreases vasoconstriction. Although the mechanism by means of which E2 modulates BK channels has not been completely described and the binding site of E2 in the BK channel has not been identified, it was demonstrated that the presence of the accessory β1 subunit is necessary for this modulatory effect (Valverde et al., 1999). Our results indicate that the β1 subunit can be expressed in a heterologous system alone, and by performing binding assays using the fluorescent complex of E2 (E2-BSA-FITC), we show that β1 is the target of E2. Using molecular dynamics, we identified the W163 residue of the β1 subunit as a possible structural component of the binding site for E2. Consistent with this result the point mutation W163I completely abolishes E2 binding. We propose that the binding site for E2 is located in the β1 subunit of the BK channel and that the W163 residue is directly involved in the binding between the BK channel and E2.

BK channels are modulated by β-subunits (β1-β4) in a tissue-specific manner. The β1- and β2-subunits increase in the apparent Ca2+ sensitivity of BK can be explained by a stabilization of the voltage sensor domain (VSD) in its active configuration. However, whether β1 modifies the number of charges associated with the voltage sensor activation is still a matter of controversy. The purpose of our study is to determine the effects of the presence of β-subunits on the gating charge in the presence of Ca2+, and to evaluate the coupling between Ca2+-binding and VSD activation. The number of gating charges per channel was measured in BK channels formed by α-subunit alone and with the different β-subunits in Ca2+-free internal solutions. The maximum gating charge displaced was obtained from the charge-voltage (Q-V) curve and the total number of channels in the patch was determined using noise analysis. Furthermore, we evaluated the effect of β-subunits on the interaction between Ca2+ sensors and VSD in different Ca2+ concentrations. We found that the total number of charges per channel was 4.4, 3.0 and 4.2 e0 for BKα, BKα/β1 and BKα/β3b channels respectively. Increasing intracellular [Ca2+] in BKα (100 µM) promotes a significant leftward shift (∼-140 mV) of the Q-V curve. The calcium effect on voltage sensor in BKα becomes apparent at [Ca2+] ≥ 1 µM (∼∆V −30 mV). However, the leftward shift of the Q-V curve in BKα/β1 channels becomes evident in the nanomolar [Ca2+] range (100-500 nM). BKα/β3b channels behave as BKα channels. We conclude that: a) β1-subunit not only modifies the resting-active equilibrium of the voltage sensor but also decreases the total number of apparent gating charges; b) there is a strong coupling between voltage and Ca2+ sensors, this coupling is increased in the presence of β1-subunit.

Connexins (Cxs) are transmembrane proteins involved in the electrical coupling of cells, the release of signaling molecules, and cell proliferation among others. These proteins are expressed in almost every tissue of the human body and mutations in these proteins are related to several hereditary diseases. Cxs monomers oligomerize in hexamers, which traffic to the plasma membrane and form connexons or hemichannels that can act as plasma membrane channels or can travel to intercellular contact zones where they form intercellular channels known as gap junctions. The opening of the Cx hemichannels in the membrane is tightly regulated by Ca2+ and membrane voltage. This regulation prevents leakage of cellular content and its malfunction may lead to pathologic conditions. In this work we studied the reciprocal regulation of Cxs hemichannels by Ca2+ and voltage. We expressed Cx46 in Xenopus laevis oocytes, and using two electrode voltage clamp, analyzed the inhibition of currents by Ca2+. We observed that Cxs inhibition by Ca2+ is voltage dependent. The apparent Ca2+ sensitivity is increased as the membrane voltage is made more negative. This change in the affinity can be explained by a stabilization of the closed state of the Cx channel by Ca2+. We propose a model in which the calcium-bonded state prevents the opening of the hemichannel. The model allows us to determine the affinity of the closed state for calcium. In agreement with the model and suggesting a stabilization of a conformational change that narrows the pore, calcium inhibits the water flux trough Cx hemichannels. This work supports the idea that Ca2+ and voltage act synergistically to promote the closing of the pore in Cx channels.