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PLoS One
2014 Jan 01;910:e109306. doi: 10.1371/journal.pone.0109306.
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Both transmembrane domains of BK β1 subunits are essential to confer the normal phenotype of β1-containing BK channels.
Kuntamallappanavar G
,
Toro L
,
Dopico AM
.
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Voltage/Ca²⁺(i)-gated, large conductance K+ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel's apparent Ca²⁺(i) sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions ("background") from β4 showed a phenotype (V(half), τ(act), τ(deact)) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β "halves" (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function.
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Figure 2. Characteristic BK current phenotype of channels made of cbv1± β1 or β4 subunits.Representative traces of macroscopic current recordings and averaged G/Gmax-V plots (B) obtained from I/O oocyte membrane patches expressing cbv1 (construct 1), cbv1+hβ1 (construct 2) or cbv1+hβ4 (construct 3); Ca2+i = 10 µM. Bar graphs show averaged Vhalf (C), activation (D) and deactivation (E) time constants (τact, τdeact, respectively) obtained for cbv1, cbv1+hβ1, and cbv1+hβ4; Ca2+i = 10 µM. *Different from cbv1 (P<0.05); #Different from cbv1+β1 (P<0.05). Error bars correspond to SEM; each point represents the average of ≥4 patches.
Figure 3. Both TMs of β1 are required for conferring the characteristic phenotype of β1-containing BK channel complexes.(A) Cartoons depicting the chimeric constructs obtained by swapping TM protein regions between hβ1 and hβ4 subunits. Regions from β1 and β4 are given in black and grey, respectively. (B) Macroscopic current recordings obtained from I/O oocyte membrane patches expressing cbv1+β1TMs4 (construct 4) and cbv1+β4TMs1 (construct 5) Ca2+i = 10 µM. (C) Averaged G/Gmax-V plots of constructs 1–5 obtained at Ca2+i = 10 µM. Averaged Vhalf (D), activation (E) and deactivation (F) time constants (τact, τdeact, respectively) from constructs 1–5 obtained at 10 M Ca2+i. *Different from cbv1 (P<0.05); #Different from cbv1+β1 (P<0.05). Error bars correspond to SEM; each point represents the average of ≥4 patches.
Figure 4. Neither TM1 or TM2 from BK β1 is sufficient to confer the characteristic phenotype of β1-containing to BK channel complexes.(A) Cartoons depicting chimeric constructs that result from swapping individual transmembrane domains (either TM1 or TM2) between hβ1 and hβ4. Regions from β1 and β4 are given in black and grey, respectively. (B) Macroscopic current recordings obtained from I/O oocyte membrane patches expressing cbv1+β4TM11 (construct 6) or cbv1+β4TM21 (construct 7); Ca2+i = 10 µM. (C) Averaged G/Gmax-V plots of cbv1, cbv1+hβ1, cbv1+hβ4, cbv1+β4TM11 and cbv1+β4TM21; Ca2+i = 10 µM. Averaged Vhalf (D), activation (E) and deactivation (F) time constants (τact, τdeact, respectively) obtained cbv1, cbv1+hβ1, cbv1+hβ4, cbv1+β4TM11 and cbv1+β4TM21; Ca2+i = 10 µM. *Different from cbv1 (P<0.05); #Different from cbv1+β1 (P<0.05). Error bars correspond to SEM; each point represents the average of ≥4 patches.
Figure 5. Physical continuity of the EC loop between TM1 and TM2 is essential to confer the characteristic phenotype of β1-containing BK channel complexes.(A) Cartoons depicting two hβ1/hβ4 chimeric constructs termed “N-half” and “C-half”, obtained by cleaving the EC loop between TM1 and TM2 in the β4TMs1 chimera. Regions of β1 and β4 are given in black and grey, respectively. When expressed together (panels C-G and main text), “N-half” and “C-half” chimera have been labeled as construct 8. (B) Western blots reflecting the surface presence of N-half and C-half, when co-expressed with cbv1, obtained by surface biotinylation of Xenopus oocytes expressing cbv1+N-half+C-half complexes. Blot image where left and right lanes contain samples from uninjected and N-half+C-half chimera-injected oocytes, respectively. (C) Representative traces of macroscopic current recordings obtained from I/O oocyte membrane patches expressing construct 8; Ca2+i = 10 µM. (D) Averaged G/Gmax-V plots from cbv1, cbv1+β1, cbv1+β4, and cbv1+construct 8; Ca2+i = 10 µM. Averaged Vhalf (E), activation (F) and deactivation (G) time constants (τact, τdeact, respectively) obtained cbv1, cbv1+β1, cbv1+β4, and cbv1+construct 8. *Different from cbv1 (P<0.05); #Different from cbv1+β1 (P<0.05). Error bars show SEM; each point represents the average of ≥4 patches.
Figure 1. Schematic structure of β1 subunit-containing BK channel.Cartoon showing a slo1-β1 subunit heterodimer. The channel-forming slo1 subunit includes transmembrane (TM) segments S0-S6 and intracellular Regulatory of Conductance for K+ (RCK) domains, these domains including distinct residues that participate in sensing changes in Ca2+
i. Four slo1 monomers assemble to render fully functional BK channels. All four types of β subunits identified so far contain a similar design that includes intracellular N- and C-terminals, two transmembrane domains (TM1 and TM2), and an EC loop. EC and IC correspond to the extracellular and intracellular sides of the membrane.
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