To quantify the modulation of KCNQ2/3 current by [Ca²⁺]_i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca²⁺ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative CaM (DN CaM). We varied [Ca²⁺]_i from < 10 to > 400 nM with ionomycin (5 µM) and either 2 mM Ca²⁺ or EGTA-buffered 0 Ca²⁺. wt CaM made KCNQ2/3 currents highly sensitive to [Ca²⁺]_i (IC₅₀ 70 ± 20 nM, maximum inhibition 73 %, n = 10). However, DN CaM rendered KCNQ2/3 currents largely [Ca²⁺]_i insensitive (maximum inhibition 8 ± 3 %, n = 10). In cells without co-transfected CaM, the Ca²⁺ sensitivity was very variable but generally weak. Co-immunoprecipitations showed binding of CaM to KCNQ3-5 that was similar in the presence of 0.5 mM Ca²⁺ or 0.5 mM EGTA. Gel-shift analyses suggested Ca²⁺-dependent CaM binding to an ‘IQ-like’ motif in the carboxy-terminus of KCNQ3. We tested whether bradykinin modulation of M-current in superior cervical ganglion (SCG) neurons uses CaM. wt or DN CaM was exogenously expressed in SCG neurons using pseudovirions. With EGFP only, the inhibition was 76 ± 9 % (n = 6); with DN CaM, it was 31 ± 10 % (n = 9), and with wt CaM, it was 33 ± 9 % (n = 8). In all three groups, muscarinic inhibition of M-current was normal. We observed similar [Ca²⁺]_i rises by bradykinin in the three groups, indicating that CaM did not affect Ca²⁺ release from stores. Finally, [Ca²⁺]_i-modulation of M-current in non-pseudovirus-infected SCG cells followed the same pattern as in CHO cells overexpressing KCNQ2/3 and wt CaM, suggesting that endogenous M-current in neurons is sensitive to [Ca²⁺]_i as well. We conclude that M-type currents are highly sensitive to [Ca²⁺]_i and that calmodulin may act as their Ca²⁺ sensor.

This work was supported by a HHMI New Faculty Startup Grant and NIH Grant NS43394.