oltage-and Ca 2ϩ -activated K ϩ channels (BK Ca ) are membrane proteins that play a fundamental role in controlling smooth muscle tone and neuronal excitability. In most of the tissues, they form a complex consisting of a pore-forming ␣ subunit and regulatory ␤ subunits. The ␣ subunit encodes for the selective pore as well as for the voltage and Ca 2ϩ -sensing structures. The BK Ca channel is a tetramer with each ␣ subunit organized in seven transmembrane domains (S0-S6) (1), a long intracellular C-terminal domain where a high-affinity Ca 2ϩ -binding site has been identified (2, 3), and an extracellular N terminus. The human isoform (hSlo), similarly to other voltagedependent ion channels, possesses a voltage sensor that is mainly represented by the S4 transmembrane domain, containing three positively charged residues (4, 5). Changes in membrane potential displace the voltage sensor and, for adequate depolarizations, the consequent conformational change sets the channel in a conducting state. The movement of the voltage sensor produces a transient current (gating current) that precedes in time and voltage the ionic current activation (6, 7). Thus, gating currents report on the rearrangement of the channel structure in a varying membrane potential but do not provide direct information regarding the motion of regions of the channel outside the voltage field. Structural changes occurring during gating have been elegantly resolved by using site-directed fluorescent labeling, a technique pioneered in the E. Isacoff laboratory (8) and applied to a variety of voltage-gated K ϩ and Na ϩ channels (9-24), ligand-gated channels (25,26), and transporters (27-31). However, nothing is known regarding the dynamical changes of BK Ca channel during gating. Using site-directed fluorescence labeling combined with the cut-open oocyte voltage clamp technique (COVG), we have resolved the conformational changes occurring in hSlo voltage-sensing region, unraveling extremely slow conformational changes not expected from gating current measurements. We have used thiol-reactive fluorescent probes [tetramethyl rhodamine-5-maleimide (TMRM) or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium methansulfonate (PyMPO)] to assess the dynamics of the S4 region conformational changes in BK Ca channels. ResultsBK Ca channels possess a functional voltage sensor as demonstrated by direct measurement of ionic and gating currents in the absence of internal Ca 2ϩ (6,7,32,33). In addition, Diaz et al. (4) have shown that mutations in the S4 segment alter the voltage dependence of hSlo channel activation. If the S4 transmembrane segment of BK Ca channels is part of the voltage-sensing machinery, conformational changes of the S4 region should share some of the features of channel-gating currents. In this study, we have investigated conformational changes of the region between the S3 and S4 transmembrane segments in the hSlo channel. The region of interest and the residues fluorescently labeled in this study are illustrated in a schematic...

The β2 subunit of the large conductance Ca2+- and voltage-activated K+ channel (BKCa) modulates a number of channel functions, such as the apparent Ca2+/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BKCa channels (hSlo), we have investigated the mechanisms of operation of the β2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BKCa channels coexpressed with the β2 subunit. The experimental observations are consistent with the view that the β2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium and that the β2-induced inactivation process does not follow a typical N-type mechanism.

The cytoplasmic side of the voltage-dependent Na ϩ channel pore is putatively formed by the S6 segments of domains I to IV. The role of amino acid residues of I-S6 and II-S6 in channel gating and local anesthetic (LA) block was investigated using the cysteine scanning mutagenesis of the rat skeletal muscle Na ϩ channel (Na v 1.4). G428C uniquely reduced sensitivity to rested state or first-pulse block by lidocaine without alterations in the voltage dependence or kinetics of gating that would otherwise account for the increase in the IC 50 for block. Mutations in I-S6 (N434C and I436C) and in II-S6 (L785C and V787C) increased sensitivity to first-pulse block by lidocaine. Enhanced inactivation accounted for the increased sensitivity of N434C to lidocaine and hastening of inactivation of I436C in the absence of drug could account for higher affinity first-pulse block. Mutations in I-S6 (I424C, I425C, and F430C) and in II-S6 (I782C and V786C) reduced the use-dependent lidocaine block. The reduction in use-dependent block of F430C was consistent with alterations in inactivation gating; the other mutants did not exhibit gating changes that could explain the reduced sensitivity to lidocaine. Therefore, several amino acids (I424, I425, G428, I782, and V786), in addition to those previously identified (Yarov-Yarovoy et al., 2002), alter the sensitivity of Na v 1.4 to lidocaine, independent of mutation-induced changes in gating. The magnitude of the change in the IC 50 values, the isoform, and LA dependence of the changes in affinity suggest that the determinants of binding in I-S6 and II-S6 are subsidiary to those in IV-S6.Voltage-dependent sodium (Na ϩ ) channels are key mediators of cellular excitability and targets of local anesthetic (LA) antiarrhythmic and anticonvulsant drugs. Each Na ϩ channel is formed by an ␣ subunit that is a large membranespanning glycoprotein composed of four homologous domains (I-IV) and, depending on tissue type, one or more smaller accessory ␤ subunits (Catterall, 2000). Each domain of the ␣ subunit contains six transmembrane segments (S1-S6). A portion of the cytoplasmic mouth of ion-conducting pore of the Na ϩ channel is thought to be formed by the carboxyterminal portion of the S6 of each domain.Important insights into the structure and function of the outer mouth of the Na ϩ channel pore have been revealed by site-directed mutagenesis (Chiamvimonvat et al

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