Low Voltage Activation of KCa1.1 Current by Cav3-KCa1.1 Complexes

<div><p></p><p>Calcium-activated potassium channels of the KCa1.1 class are known to regulate repolarization of action potential discharge through a molecular association with high voltage-activated calcium channels. The current study examined the potential for low voltage-activated Cav3 (T-type) calcium channels to interact with KCa1.1 when expressed in tsA-201 cells and in rat medial vestibular neurons (MVN) <i>in vitro</i>. Expression of the channel α-subunits alone in tsA-201 cells was sufficient to enable Cav3 activation of KCa1.1 current. Cav3 calcium influx induced a 50 mV negative shift in KCa1.1 voltage for activation, an interaction that was blocked by Cav3 or KCa1.1 channel blockers, or high internal EGTA. Cav3 and KCa1.1 channels coimmunoprecipitated from lysates of either tsA-201 cells or rat brain, with Cav3 channels associating with the transmembrane S0 segment of the KCa1.1 N-terminus. KCa1.1 channel activation was closely aligned with Cav3 calcium conductance in that KCa1.1 current shared the same low voltage dependence of Cav3 activation, and was blocked by voltage-dependent inactivation of Cav3 channels or by coexpressing a non calcium-conducting Cav3 channel pore mutant. The Cav3-KCa1.1 interaction was found to function highly effectively in a subset of MVN neurons by activating near –50 mV to contribute to spike repolarization and gain of firing. Modelling data indicate that multiple neighboring Cav3-KCa1.1 complexes must act cooperatively to raise calcium to sufficiently high levels to permit KCa1.1 activation. Together the results identify a novel Cav3-KCa1.1 signaling complex where Cav3-mediated calcium entry enables KCa1.1 activation over a wide range of membrane potentials according to the unique voltage profile of Cav3 calcium channels, greatly extending the roles for KCa1.1 potassium channels in controlling membrane excitability.</p></div

Cav3 calcium activation of KCa1.1 modulates repolarizing responses and gain of spike firing.

<p><i>A,</i> Representative superimposed recordings of spike discharge before and after perfusion of 1 µM mibefradil (Mib) to block the Cav3-KCa1.1 interaction. Mibefradil slows that rate of repolarization (<i>inset</i>) and reduces the AHP. <i>B,</i> Representative examples of spike firing during square wave current pulse injections before and after 1 µM mibefradil showing an increase in the rate of firing. <i>C</i>, Current-frequency plot for the cell in (<i>B</i>) reveals an increase in the gain of firing frequency in mibefradil. <i>D</i>, Bar plots of the mean rate of spike repolarization, AHP amplitude, and firing rate gain on current-frequency plots before and after mibefradil. Membrane potential was set through bias current injection to ∼−74 mV to subdue baseline tonic firing of MVN cells and spike properties were measured near threshold firing level evoked by square wave current injections. Sample sizes are shown in brackets in (<i>D</i>).</p

Cav3.2 and KCa1.1 channels associate in rat brain.

<p><i>A,</i> Dual label immunocytochemistry confirms Cav3.2 and KCa1.1 protein expression in MVN cells with a similar distribution pattern at the level of the soma and at least the proximal dendritic region. <i>B,</i> Control image upon omission of primary antibodies. <i>C,</i> Western blots showing coimmunoprecipitation of Cav3.2 and KCa1.1 protein from lysates of rat brain (<i>n</i> = 4), cerebellum (<i>n</i> = 6), and brain stem (<i>n</i> = 5), with a corresponding label for KCa1.1 in lysates from each region. In lane 1, channel complexes were immunoprecipitated using the Cav3.2 antibody, in lane 2 the precipitating antibody was omitted, and lane 3 corresponds to lysate. All Western blots were probed with anti-KCa1.1. Scale bar in (<i>A, B</i>) = 20 µm.</p

The Cav3-KCa1.1 interaction can activate KCa1.1 in the subthreshold voltage range.

<p>Representative whole-cell currents in MVN cells during a ramp command from −100 mV to −30 mV in the presence of 30 µM Cd²⁺ to block HVA calcium channels. <i>Dashed lines</i> indicate onset of the voltage ramp command. The relative time during the ramp at which different voltages are attained are shown on a voltage scale below. <i>A,</i> Outward current apparent in control recordings (<i>black</i> trace) is reduced by the KCa1.1 channel blocker paxilline (1 µM) (<i>red</i> trace) and isolated below as a difference current (<i>a-b, blue</i> trace). <i>B,</i> Recordings from a separate cell under the same conditions as in (<i>A</i>). Perfusing 1 µM mibefradil (Mib, <i>red</i> trace) blocks both inward and outward currents apparent in control recordings (<i>black</i> trace), with the difference current indicated below (<i>a-b</i>, <i>blue</i> trace). <i>C</i>, Superimposed average records of currents sensitive to paxilline (<i>black</i> trace) or mibefradil (<i>green</i> trace). Current recordings in (<i>C</i>) are average traces with SEM shown by shaded regions. Sample sizes are shown in brackets.</p

Effects of intracellular calcium buffering on KCa1.1 channel recruitment by Cav3 calcium influx.

<p><i>A</i>, Shown are whole-cell recordings from transiently transfected tsA-201 cells, demonstrating the effects of intracellular calcium buffering by 10 mM of EGTA or BAPTA on KCa1.1 current when coexpressed with Cav3.2 channels. <i>B</i>, Bar plots indicating the fold change in peak KCa1.1 current (P2/P1) under the indicated conditions of internal calcium chelation when coexpressed with Cav3.2 channels. Calcium entry via Ca_v3.2 channels increases KCa1.1 channel activity in the presence of internal 0.1 mM EGTA or 0.1 mM BAPTA, but not in the presence of 10 mM of either calcium chelator, or with alternate expression of the nonconducting Ca_v3.2^pm. Sample sizes are shown in brackets.</p

Ca_v3.2 channels interact with the N-terminal transmembrane region of KCa1.1 channels.

<p><i>A</i>, A schematic drawing of the α-subunit of KCa1.1 with the N-terminal segment. Two alternate N-terminal splice variant sequences are shown below the diagram (N+S0, aN+S0) with S0 designating the transmembrane component of the N-terminal region. Region of overlap between KCa1.1 N+S0 and aN+S0 are shown in <i>grey shading</i>. Constructs tested include full length KCa1.1, KCa1.1 N+S0, KCa1.1 aN+S0, KCa1.1 N-terminus, KCa1.1 without C-terminus (KCa1.1ΔC-term), and KCa1.1 C-terminus (<i>n</i> = 4). <i>B</i>, <i>Left column</i>, Western blots from lysates of tSA-201 cells indicating that Ca_v3.2 protein coimmunoprecipitates with full length KCa1.1, KCa1.1ΔC-term, or KCa1.1 N+S0. <i>Right column</i>, Cav3.2 channels coimmunoprecipitate with KCa1.1 aN+S0 but not with the KCa1.1 N-terminus. A weak binding was also observed with KCa1.1 C-terminus and Cav3.2. In both panels B and C the first lane corresponds to an immunoprecipitation conducted with a polyclonal Cav3.2 antibody. In the second lane, the precipitating antibody was omitted (i.e., bead only control), and the third lane corresponds to tsA-201 cell lysate. In each case, Western blots were probed with an anti-myc antibody to detect myc-tagged KCa1.1 channels or their fragments. Sample sizes are shown in brackets.</p

KCa1.1 channels are regulated by Cav3 calcium influx in MVN neurons.

<p><i>A,</i> Whole-cell recording of Ni²⁺ (300 µM) -sensitive Cav3 calcium current in the presence of 30 µM Cd²⁺ to block HVA calcium channels. At <i>right</i> is the current-voltage relationship for 1 µM mibefradil (Mib)- or Ni²⁺-sensitive currents (data pooled), revealing a LVA inward calcium current that peaks at −40 mV, and progressive activation of outward current at higher membrane voltages. <i>B,</i> Representative recordings of outward current sensitive to 1 µM paxilline (Pax) or 1 mM TEA to block KCa1.1 channels, or 1 µM mibefradil or 300 µM Ni²⁺ to block Cav3 channels reveal a similar rapid peak and decaying outward current. <i>C,</i> Bar plots of peak outward current evoked by a step from −100 mV to +40 mV. The average result of applying each of 1 µM mibefradil, 300 µM Ni²⁺, 1 mM TEA, 1 µM paxilline, or 10 mM internal EGTA is compared to the extent of KCa1.1 block by inactivating Cav3 channels by a prestep to −30 mV. Mean values were statistically different from original control values, but not from each other (one-way ANOVA F(5,47) = 1.63, <i>P</i> = 0.17). <i>D</i>, Representative outward current evoked by a step from −100 mV to +40 mV when preceded by preconditioning steps from −70 mV to −20 mV to evoke Cav3 channel inactivation. Shown are traces in control medium (<i>a</i>), after perfusing 300 µM Ni²⁺ (<i>b</i>), and difference currents (<i>a–b</i>). <i>Inset</i> shows an expanded view of the Cav3 current activated by an initial prestep to −30 mV. <i>E,</i> Mean plots of the voltage-dependence of outward current evoked from −100 mV to the indicated potentials and sensitive to each of the indicated blockers. Sample sizes in (<i>A</i>, <i>C</i> and <i>E</i>) are shown in brackets and capacitance artefacts in (<i>A</i>) were reduced digitally for display purposes.</p

Cav3.2 channels increase KCa1.1 current by shifting activation to hyperpolarized potentials.

<p>Shown are whole-cell patch clamp recordings from tsA-201 cells transiently transfected with Cav3.2 or KCa1.1 cDNA. <i>A,</i> Representative KCa1.1 currents evoked with or without coexpression of Cav3.2 channels. KCa1.1 activation was tested by 250 ms steps to +40 mV (P1, P2) and the P2 pulse immediately preceded by a 50 ms test pulse to −30 mV (2 ms return to −100 mV) to maximally activate Cav3 current. <i>B,</i> Bar plots showing the fractional change (P2/P1) in KCa1.1 current elicited by the protocol in (<i>A</i>) and with Cav3.2 channels substituted with a Cav3.2 pore mutant (Ca_v3.2^pm) that does not conduct calcium. <i>C,</i> Current-voltage plots indicating that a pre-pulse to −30 mV to activate Cav3 current (see <i>inset</i>) significantly shifts KCa1.1 activation to more hyperpolarized potentials. KCa1.1 activation is unaltered when coexpressed with Ca_v3.2pm. <i>D,</i> Plots of the P2/P1 ratio of KCa1.1 current evoked at +40 mV in a tsA-201 cell as a function of the voltage of a 50 ms pre-pulse command delivered in 10 mV increments from −90 mV to +20 mV (see <i>inset</i>). Note the close correspondence in voltage-dependence and magnitude of Cav3.2 and KCa1.1 currents. <i>E,</i> Time dependence of KCa1.1 activation from the effects of a pre-pulse to −30 mV (time 0) with test pulses to +40 mV (see <i>inset</i>) delivered at the indicated time intervals in cells with or without coexpression of Cav3.2 channels. Capacitance artefacts in (<i>A</i>) were digitally reduced for display purposes. Sample sizes are shown in brackets.</p

Model of Cav3 calcium channel domain and interaction with KCa1.1 channels.

<p>A calcium diffusion model is used to simulate the interaction between a Cav3 calcium source and a KCa1.1 channel located within its nanodomain (20 nm). Cav3.2 and KCa1.1 properties are based on reported values in the presence of physiological levels of [Ca]_o. <i>A,</i> Activation curves for the KCa1.1 model based on the increase of [Ca]_i when 1, 2, 4, or 8 Cav3 channels are positioned within 20 nm (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061844#s2" target="_blank">Methods</a>). A single Cav3 channel causes no activation of a KCa1.1 channel below −30 mV and only minimal activation at high voltages. Activation of KCa1.1 at low voltages (−50 mV) is only attained with 4 or 8 Cav3 channels. <i>B,</i> Activation time courses during voltage steps as in (<i>A</i>) for models with either 1 or 4 Cav3 channels positioned within 20 nm of KCa1.1. KCa1.1 activation is transient, tracking the Cav3-mediated calcium influx, but with much higher probability when 4 Cav3 channels contribute. <i>C, D,</i> Schematic diagrams of a proposed model for Cav3 activation of KCa1.1 channels. In the case of a single Cav3 channel (<i>C</i>) a small calcium domain provides only a low probability of generating the increase in [Ca]_i necessary to activate KCa1.1 channels. By comparison, summation of calcium domains generated by several neighboring Cav3/KCa1.1 channel complexes (<i>D</i>) is sufficient to activate KCa1.1. Such a contribution of more distant Cav3 channels would then be consistent with the EGTA sensitivity observed in our experiments.</p

RT-PCR analysis of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Schematic representation of the location of PCR primers used. Primers RR44, 45, and 46 were used for RT-PCR reactions; primers RR50, 51, 52, and 53 were used for genomic PCR reactions. B. Agarose gel depicting RT-PCR reaction products for mRNA isolated from <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice. Regardless of the primer pair used, only a single band is detected using mRNA from <i>Cacna1f^wt</i> mice. Using mRNA from <i>Cacna1f^nob2</i> mice, however, two bands are visible (see arrows). The relative intensities of the fluorescence signals indicate that the larger-M_r band accounts for ∼90%, and the smaller-M_r band for ∼10%, of the total mRNA.</p