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.
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.